Emerging Model Organisms

Paramecium tetraurelia: The Renaissance of an Early Unicellular Model Janine Beisson,1 Mireille Bétermier,1 Marie-Hélène Bré,2 Jean Cohen,1 Sandra Duharcourt,3 Laurent Duret,4 Ching Kung,5 Sophie Malinsky,3 Eric Meyer,3,7 John R. Preer Jr,6 and Linda Sperling1 1

Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, FRE3144, F-91198 Gif-sur-Yvette, France Laboratoire de Biologie Cellulaire 4, Centre National de la Recherche Scientifique, UMR 8080, Université Paris-Sud, 91405 Orsay Cedex, France 3 Laboratoire de Génétique Moléculaire, Centre National de la Recherche Scientifique, UMR 8541, École Normale Supérieure, F-75230 Paris, France 4 Laboratoire de Biométrie et Biologie Évolutive, Centre National de la Recherche Scientifique, UMR 5558, Université Lyon 1, F-69622, Villeurbanne, France 5 Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin-Madison, WI 53706, USA 6 Department of Biology, Indiana University, Bloomington, IN 47405-3700, USA 2

INTRODUCTION Paramecium tetraurelia is a widely distributed, free-living unicellular organism that feeds on bacteria and can easily be cultured in the laboratory. Its position within the phylum Ciliophora, remote from the most commonly used models, offers an interesting perspective on the basic cellular and molecular processes of eukaryotic life. Its large size and complex cellular organization facilitate morphogenetic studies of conserved structures, such as cilia and basal bodies, as well as electrophysiological studies of swimming behavior. Like all ciliates, P. tetraurelia contains two distinct types of nuclei, the germline micronucleus (MIC) and the somatic macronucleus (MAC), which differentiate from copies of the zygotic nucleus after fertilization. The sexual cycle can be managed by controlling food uptake, allowing the study of a developmentally regulated differentiation program in synchronous cultures. Spectacular genome rearrangements occur during the development of the somatic macronucleus. Their epigenetic control by RNA-mediated homology-dependent mechanisms, which might underlie long-known cases of non-Mendelian inheritance, provides evolutionary insight into the diversity of small RNA pathways involved in genome regulation. Being endowed with two alternative modes of sexual reproduction (conjugation and autogamy), P. tetraurelia is ideally suited for genetic analyses, and the recent sequencing of its macronuclear genome revealed one of the largest numbers of genes in any eukaryote. Together with the development of new molecular techniques, including complementation cloning and an easily implemented technique for reverse genetics based on RNA interference (RNAi), these features make P. tetraurelia a very attractive unicellular model.

RELATED INFORMATION More extensive information about Paramecium biology can be found in the ParameciumDB wiki: http://paramecium.cgm.cnrs-gif.fr/parawiki/Paramecium_Biology.

BACKGROUND INFORMATION Paramecium was among the first aquatic microorganisms observed after the invention of the microscope. In 1773, the first species of the genus was named Paramecium aurelia using the rules of Linnean

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Corresponding author ([email protected]). Cite as: Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.emo140

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classification; P. aurelia has since been shown to consist of a group of 15 related subspecies (Sonneborn 1974; Catania et al. 2009). There are actually 14 different Paramecium species (or groups of sibling species, such as the aurelia complex) distinguishable by morphological or physiological differences, forming a distinct monophyletic group of ciliates within the subphylum Intramacronucleata and the class Oligohymenophorea (Strüder-Kypke et al. 2000). These large (~100-250 mm in length) free-living unicellular organisms are distributed widely in fresh waters, where they feed on bacteria, yeast, or algae (for general reviews on Paramecium biology, see Sonneborn 1974; Beale and Preer 2008). Most species have been found on several continents and some are clearly cosmopolitan. Clonal cell lines can be established easily by single cell isolation and can be grown in bacterized grass infusion medium for a large number of vegetative divisions (~200 for P. tetraurelia) before entering senescence. Sexual reproduction is triggered by starvation and therefore can be induced or prevented as needed by controlling the availability of food (Sonneborn 1950). Like all ciliates, Paramecium has a highly complex cellular structure (Fig. 1). Thousands of cilia anchored at the cell surface organize the cytoskeleton and control cell movements and feeding activity, as well as reactivity to sexual partners and to the environment. Specialized organelles fulfill distinct physiological functions, such as phagocytosis, digestion, secretion, excretion, contraction, and osmoregulation. One of the main biological innovations of ciliates is their nuclear dualism. In each sexual generation, two kinds of nuclei develop from copies of the zygotic nucleus: The polyploid MAC is a somatic nucleus that contains a genome streamlined for gene expression, whereas the diploid MIC remains transcriptionally silent and only serves germline functions, transmitting genetic information to the next generation through meiosis (Fig. 2). This offers a unique opportunity to study developmentally regulated germline/somatic differentiation in a unicellular model. Among ciliates, P. aurelia species are ideally suited for genetic analyses because of their two alternative modes of sexual reproduction. Conjugation, the reciprocal fertilization of two cells of complementary mating types, yields a pair of F1 cells that are always genetically identical, but develop within different parental cell structures and cytoplasms, allowing the easy detection of non-Mendelian inheritance; autogamy is a useful self-fertilization process that results in an entirely homozygous genome in just one generation (Fig. 3). Paramecium was developed as a model system by the American biologists H. S. Jennings (18681947) and T. M. Sonneborn (1906-1981). Studies in the late 19th and early 20th centuries led to a precise knowledge of its cytology and life cycle. Focusing on P. aurelia, Sonneborn established the rules governing conjugation between complementary mating types (Sonneborn 1937) and developed methods for crossing lines of different phenotypes and maintaining homozygous strains (Sonneborn 1950). His studies showed that the different “varieties” of P. aurelia (initially designated as Variety or Syngen 1, 2, 3, etc.) represented 15 distinct, non-cross-fertile sibling species that eventually were named Paramecium primaurelia, Paramecium biaurelia, etc. The first phenotypic variations subjected to breeding analysis in P. tetraurelia revealed complex relationships between nuclear genes, cytoplasm, and environment, contrasting with genetic studies in other models. In the mid-1950s, Paramecium thus appeared to be an interesting but somewhat exotic organism. Nevertheless, Sonneborn’s discoveries attracted a number of other researchers who propagated the model. Using genetic approaches and selection of mutants after mutagenesis, these groups explored biological problems

FIGURE 1. Phase contrast image of P. tetraurelia. Ci: cilia; CVC: contractile vacuoles; DV: digestive vacuoles; MAC: macronucleus; MIC: micronuclei; OA: oral apparatus; PM: plasma membrane; Tr: trichocysts. The anteroposterior polarity (A-P) is indicated. www.cshprotocols.org

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FIGURE 2. Vegetative and sexual phases of the Paramecium life cycle. The vegetative cell cycle (green arrows) consists of the mitotic division of the two MIC and the nonmitotic division of the macronucleus MAC. Nuclear reorganization events occurring during autogamy (purple arrows) include meiosis of the two diploid MIC, degeneration of seven of the eight haploid products and additional division of the single surviving one, karyogamy, two mitotic divisions of the diploid zygotic nucleus, and development of two new MAC, which segregate without division to the two daughter cells at the first cellular division. (For color figure, see doi: 10.1101/ pdb.emo140 online at www.cshprotocols.org.)

such as bacterial endosymbionts, swimming behavior and electrophysiology, basal-body duplication and “structural inheritance,” non-actin-based contractility, exocytosis, mitochondrial genetics and nucleomitochondrial interactions, post-translational tubulin modifications, variant genetic codes, programmed genome rearrangements (and their epigenetic regulation by noncoding RNAs), RNAi, and genome evolution. Since the cloning of the first genes some 20 yr ago, new methods have been developed for immunocytochemistry, transformation of the MAC with cloned genes, complementation cloning, and RNAi-based reverse genetics. With the complete sequencing of its macronuclear genome (Aury et al. 2006), P. tetraurelia has once again emerged as a promising model organism.

SOURCES AND HUSBANDRY Standard procedures for maintaining P. tetraurelia lines of known clonal age through weekly cycles of vegetative growth and autogamy, their expansion by mass culture, and the maintenance of stocks were established by Sonneborn (1950). These basic protocols are described in Maintaining Clonal Paramecium tetraurelia Cell Lines of Controlled Age through Daily Reisolation (Beisson et al. 2010a) and Mass Culture of Paramecium tetraurelia (Beisson et al. 2010b). Geographic isolates, wild-type reference strains and numerous mutant strains are maintained in liquid culture at 13°C-18°C or, less frequently, frozen using special equipment and stored in liquid

FIGURE 3. Genetic analyses of Mendelian and non-Mendelian inheritance. (Left) Mendelian segregation of a pair of alleles. Conjugation between two cells homozygous for different alleles at one locus (+/+ and m/m) yields genetically identical heterozygous F1 progeny; autogamy of these F1 clones results in entirely homozygous F2 clones that have a 50% chance of retaining each of the parental alleles. (Center) Cytoplasmically inherited characters (e.g., characters determined by the mitochondrial genome) are inherited as depicted; very little cytoplasm is exchanged between the two mates during conjugation. (Right) Mating type inheritance follows cytoplasmic lines rather than nuclear genes, even though the mating type is determined by the mature MAC; this is more properly called maternal inheritance. (For color figure, see doi: 10.1101/pdb.emo140 online at www.cshprotocols.org.)

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nitrogen. Sonneborn (1974) published a catalog of all available P. aurelia strains, and many of the stocks corresponding to natural populations are available through the American Type Culture Collection or academic laboratories that maintain such collections (e.g., the laboratories of Ewa Przybo, Alexey Potekhin, and Michael Lynch). Similarly, collections of mutant strains have been established by the laboratories of Ching Kung (behavioral mutants), Jim Berger (cell cycle mutants), and Janine Beisson (primarily mutants affecting cell shape, exocytosis of secretory granules, and matingtype determination). Thanks to the occurrence of autogamy, each stock is genetically homogeneous and has an entirely homozygous germline genome. The Beisson collection is actively maintained; a catalog is available online (http://paramecium.cgm.cnrs-gif.fr/) and there are plans to include information about strains maintained in other academic collections.

RELATED SPECIES Among the 15 sibling subspecies of the P. aurelia group, P. tetraurelia has been used most extensively as a model system, although important work on the genetics and molecular genetics of surface antigens was carried out in P. primaurelia (for review, see Caron and Meyer 1989). Among the 14 species making up the Paramecium genus overall, three other species have been the object of sustained investigations. In Paramecium caudatum, studies focused on behavioral genetics (Takahashi and Naitoh 1978), conjugation, phagocytosis, bacterial endosymbiosis, mating-type, and nuclear differentiation, as well as gravitaxis and thigmotaxis. Paramecium bursaria, with its symbiotic green algae of the genus Chlorella, provides a pertinent model to study ciliate/algae symbiosis. The largest species, Paramecium multimicronucleatum, has been used primarily for ultrastructural cytological studies.

USES OF THE P. TETRAURELIA MODEL SYSTEM Developmentally Regulated Genome Rearrangements

During sexual reproduction, the parental MAC is lost and replaced by a new one that develops from a copy of the zygotic nucleus formed by meiosis of the MIC and karyogamy (Fig. 2). In addition to massive amplification of the diploid genome to ~800n, MAC development involves two types of reproducible DNA elimination events that begin after a few initial rounds of endoreplication (Fig. 4; for review, see Bétermier 2004). By managing the life cycle, it is possible to induce these extensive rearrangements synchronously in mass cultures undergoing either conjugation or autogamy. The system thus provides an ideal model for studying the molecular mechanisms of DNA recombination. Recent progress has been made toward unraveling the mechanisms underlying the precise excision of internal eliminated sequences (IES). These short (26-882 base pairs [bp]) noncoding sequences, which can be described as DNA “introns,” were discovered when a MIC locus was

FIGURE 4. Genome rearrangements during MAC development. DNA amplification (2n to ~800n), precise excision of IES, and imprecise elimination of repeated sequences are shown schematically. (For color figure, see doi: 10.1101/pdb.emo140 online at www.cshprotocols.org.) www.cshprotocols.org

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sequenced and compared to its MAC counterpart (Preer et al. 1992). They are found throughout the germline genome and are invariably flanked by two 5′-TA-3′ dinucleotide repeats, one of which is left in the MAC sequence after excision. IES excision is initiated by double-stranded cleavage at both ends, centered on each TA. Although such precision is essential for the reconstitution of functional genes, IES excision is not perfectly efficient, and unexcised or aberrantly excised IES are occasionally found in one or more of the MAC copies (Duret et al. 2008). Recent work has identified some of the proteins participating in the reaction, including a domesticated transposase (Baudry et al. 2009) and DNA repair proteins (A Kapusta, A Matsuda, S Malinsky, A Silve, A Marmignon, C Baudry, E Meyer, J Forney, and M Bétermier, in prep.; S Malinsky, A.Gratias, O Garnier, A Le Mouël, S Duharcourt, M Bétermier, and E Meyer, in prep.). P. tetraurelia is thus an attractive alternative to vertebrate V(D)J recombination to study programmed genome rearrangements, double-strand break repair, and the role of transposable elements in the evolution of such systems. Imprecise elimination of ~100-200 regions of the germline genome (up to several kilobases in length) is associated with chromosome fragmentation. The resulting MAC chromosome ends show reproducible heterogeneity within each developing MAC because of variability both in the exact boundaries of deleted segments and in the number of G4T2 or G3T3 telomeric repeats that are subsequently added de novo at broken ends. At some loci, imprecise elimination can alternatively lead to internal deletions of variable lengths. Imprecisely eliminated loci each contain repeated DNA sequences (Forney and Blackburn 1988; Le Mouël et al. 2003). Transposons and minisatellites are virtually absent from the MAC genome, although it is unclear whether all copies are removed in an imprecise manner. Homology-Dependent Regulation of Genome Rearrangements by Noncoding RNAs

The means by which Paramecium cells specifically recognize such a large number of different MICspecific sequences has long been a subject for study; the weak consensus signal at IES ends clearly is not sufficient to determine the excision pattern across the whole genome. Rearrangements are partially regulated epigenetically by maternal effects (for review, see Meyer and Chalker 2007; Duharcourt et al. 2009). Such programming might involve a homology-dependent, trans-nuclear comparison of the MIC genome to be rearranged with the previously rearranged version contained in the maternal MAC, still present in the cytoplasm at that stage. Recent data suggest that a natural genomic subtraction between maternal MIC and MAC genomes is mediated by two types of noncoding RNA molecules: a special class of developmentally regulated small RNAs from the MIC and longer, noncoding transcripts produced by the MAC (Lepère et al. 2008). During early meiosis of the MIC, a specialized small RNA pathway produces massive amounts of ~25-nt scan RNAs (scnRNAs) from much of the germline genome (Lepère et al. 2009). ScnRNAs that match perfectly to longer noncoding transcripts produced by the rearranged genome of the maternal MAC would be degraded selectively or sequestered, resulting in the selection of MIC-specific scnRNAs. Once selected by this “scanning” procedure, MIC-specific scnRNAs would be re-exported to the developing zygotic MAC to target the elimination of homologous sequences. This could occur via scnRNA-induced deposition of yet uncharacterized epigenetic modifications on homologous genomic sequences. Further work will determine the extent of functional similarities between ciliate scnRNAs and metazoan Piwi-interacting RNAs, which are also produced specifically by the germline during meiosis and have been implicated in the control of transposable elements. The genome-wide comparison of transcripts from different nuclei might also shed light on RNA-mediated epigenetic processes in other eukaryotes. RNAi

The P. tetraurelia genome contains a large number of genes typically involved in RNAi, making this organism an interesting model to study the functional specialization of small RNA pathways. At present, microRNAs have not been reported in Paramecium or any other ciliate. However, in addition to the meiosis-specific scnRNA pathway, an entirely distinct set of small RNAs and proteins can mediate experimentally induced homology-dependent gene silencing throughout the life cycle (Lepère et al. 2009). Both transgene- and dsRNA-induced silencing correlate with accumulation of ~23-nt siRNAs (Garnier et al. 2004; Nowacki et al. 2005), the biogenesis of which involves the constitutively expressed DCR1 gene. Interestingly, there appear to be different subclasses of small interfering RNAs (siRNAs) associated with dsRNA-induced silencing: primary siRNAs cut by Dcr1 from both strands of the inducing dsRNA, and secondary siRNAs, all antisense to the targeted gene, copied by

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RNA-dependent RNA polymerases from the targeted endogenous mRNA (Lepère et al. 2009). Recent work has confirmed the role of these polymerases in Paramecium RNAi and of different proteins involved in transgene-induced versus dsRNA-induced silencing (S Marker, A Le Mouël, E Meyer, and M Simon, in prep.). Similarly, distinct but overlapping subsets of Piwi proteins mediate silencing induced by these two methods (K Bouhouche, J-F Goût, A Kapusta, M Bétermier, and E Meyer, in prep.). Genome Evolution

The sexually transmitted MIC genome, comprising some 50-60 small chromosomes, is currently being sequenced; a complete description of MIC-limited sequences (e.g., IES, transposons) and centromeres is not yet available. Nevertheless, the sequencing of the ~200 chromosomes comprising the rearranged MAC reveals some of the selective pressures shaping gene repertoires and intron sequences during the evolution of eukaryotic genomes. The 72-Mb MAC genome is very compact, containing 78% of the coding sequences; intergenic regions average ~352 bp (Zagulski et al. 2004; Aury et al. 2006). The genetic code of Paramecium was the first nuclear code shown to be nonstandard (Caron and Meyer 1985; Preer et al. 1985): The only stop codon is TGA, whereas TAA and TAG code for glutamine. With ~40,000 annotated protein-coding genes (almost twice as many as in mammals), P. tetraurelia is one of the most gene-rich organisms known to date (Aury et al. 2006). This is the consequence of three successive whole-genome duplications (WGD). The oldest WGD probably occurred before the divergence between the Paramecium and Tetrahymena lineages; the most recent one appears to coincide with the radiation of the P. aurelia group of species. After a WGD, duplicated chromosome pairs diverge progressively by point mutations and rearrangements. Many duplicated genes are pseudogenized or lost, making ancient WGDs difficult to recognize. The P. tetraurelia model allows the fate of duplicated genes to be studied over different time scales. Although most duplicate genes ultimately are lost, highly expressed genes and genes encoding subunits of protein complexes are much more frequently retained in two copies than the average. Thus, in the short term, natural selection appears to favor the retention of gene duplicates because of constraints on gene dosage to achieve optimal expression levels (Aury et al. 2006). In the long term, some gene duplicates are retained because they evolve toward different functions (e.g., the actin gene family; Sehring et al. 2007). Gene losses occurring independently in different populations, therefore, likely lead to rapid reproductive isolation between populations having lost different copies of each ancestral gene (Scannell et al. 2006). The explosion of speciation events that gave rise to the P. aurelia complex could have been a direct consequence of the most recent WGD (Aury et al. 2006). Analyzing gene loss patterns in different subspecies of the P. aurelia group could allow testing of the impact of WGDs on speciation. Analysis of the distribution of intron size in the P. tetraurelia genome can also contribute to our knowledge of eukaryotic gene evolution. Because the introns of the P. tetraurelia genes are extremely short (~25 bp), it was possible to clearly visualize a striking deficit of introns whose size is a multiple of three, specifically among the subset of introns that do not contain stop codons in the reading frame of the upstream exon ( Jaillon et al. 2008). This could reflect selective pressures to ensure that unspliced mRNAs will contain premature stop codons. Such aberrant mRNAs are then degraded, avoiding the expression of functionless and possibly toxic truncated proteins. Interestingly, this constraint is not restricted to ciliates, but is observed in all other intron-rich eukaryotes (plants, fungi, and animals). Thus, eukaryotic cells universally rely on translation to control the accuracy of pre-mRNA splicing (Jaillon et al. 2008). Symbionts

Although the first symbiont recognized in Paramecium was the free-living alga Chlorella in P. bursaria, most symbionts are bacteria (for review, see Görtz 2006; Beale and Preer 2008); the majority are obligate symbionts. Although most are found in the cytoplasm, others are restricted to the nuclei. Interest in symbionts first centered on the “kappa” cytoplasmic particles of P. tetraurelia because of early evidence that their maintenance during vegetative growth depends on both cytoplasmic and nuclear factors. Although transmission of the bacteria themselves to daughter cells is clearly cytoplasmic, their multiplication also involves the function of a nuclear gene, called K, for which a nonpermissive allele was identified. Symbiont-bearing strains of Paramecium called “killers” liberate a specific toxin into the medium that affects “sensitive” paramecia lacking the symbionts. Each symbiont-bearing strain is resistant to

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the specific toxin that it produces. Paramecia can be freed of symbionts by various means. When the symbionts are lost, the strains fail to kill. However, they also lose their resistance to their specific toxin; this loss of immunity has not yet been explained in molecular terms. Exocytosis

Paramecium has a prominent secretory pathway that makes it a suitable model to study regulated secretion. The Paramecium cell surface is underlined by a layer of trichocysts: voluminous secretory granules 2-3 µm in length attached to the plasma membrane that release their contents into the external medium in response to various stimuli. Trichocysts contain a crystalline protein matrix that expands longitudinally approximately eightfold when the granule membrane fuses with the plasma membrane and the matrix contacts the medium, literally shooting itself out of the cell. Trichocyst matrix proteins (TMPs), encoded by a large family of co-expressed genes, share a common protein fold despite their different primary sequences (Adoutte et al. 1984; Gautier et al. 1994). Trichocyst biogenesis involves accumulation of the TMPs in post-Golgi vesicles, protein cleavage into two mature polypeptides and crystallization (Gautier et al. 1994). Once organelle biogenesis is complete, the mature trichocyst migrates to the surface and its tip attaches to the plasma membrane. During docking, morphogenesis occurs within the plasma membrane, transforming intramembranous arrays and assembling intramembranous particles (Beisson et al. 1976). Trichocyst biogenesis and exocytosis is dispensable for Paramecium in laboratory cultures and exocytotic capacity is easy to monitor. Mutants defective in each step of the secretory process have been obtained (Cohen and Beisson 1980); nondischarge (nd) mutants deficient in the last step of membrane fusion have been used to decipher the process of membrane fusion (Bonnemain et al. 1992). Other studies revealed a novel membrane signaling pathway from inside the vesicles that might be shared by secretory pathways in other cell types (Froissard et al. 2004). Basal Bodies, Cortical Inheritance, and Cilia

Basal bodies anchored at the cell membrane and cilia play central functions in cell biology and development of metazoa. These organelles appeared with the first eukaryote and have been conserved structurally and functionally from protist to man. In Paramecium, ~4000 basal bodies anchored in the cortex organize the cytoskeleton and determine cell shape; they are aligned in parallel rows forming a polarized pattern with right/left and anteroposterior asymmetries. During division, duplication of basal bodies proceeds through a complex spatiotemporal pattern yielding two daughter cells identical to their parent (Iftode et al. 1989). Nevertheless, cortical mutations (obtained by accidental or experimental addition or inversion of pieces of cortex) are maintained through cell divisions. Genetic analysis revealed a new type of non-Mendelian inheritance, called “cortical inheritance.” Such variants demonstrate the role of preexisting structures in guiding assembly of new ones (for review, see Beisson 2007). Reverse genetics have allowed existing questions concerning the biogenesis and function of basal bodies and cilia to be addressed. These studies benefit from (1) precise knowledge of the pattern of basal body duplication, as well as of the organization, dynamics, and biochemical composition of the cortical cytoskeletal elements dependent on, or nucleated by, the basal bodies; (2) immunocytochemical tools and the ability to tag proteins by green fluorescent protein (GFP) fusion; (3) the repetitive and polarized organization of the cortex, facilitating orientation of thin sections; (4) the large size of the cells, allowing single-cell observation; and (5) the efficiency of RNAi by feeding, which allows cytological analysis of the silenced phenotype by the first division after treatment. These experimental advantages make it worthwhile to examine in P. tetraurelia the function of conserved basal body components studied in other models. Like basal bodies, cilia have a long history in P. tetraurelia. As in other organisms, cilia fulfill both sensory and motility functions, resulting in easily observable characteristic swimming behaviors. Cilia also function in phagocytosis and cytokinesis, two processes that provide further criteria for detecting ciliary dysfunction. Genomic and proteomic data have confirmed the high level of conservation of ciliary proteins (Arnaiz et al. 2010) and preliminary experiments of RNAi-mediated inactivation of P. tetraurelia orthologs of human ciliary genes showed dramatic changes in ciliogenesis and ciliary beating. Furthermore, it appears possible to rescue the defective inactivated phenotype by the human ortholog. Both the biological properties and the range of available methods thus make it easy to approach the function of any chosen ciliary gene, in particular genes suspected to be involved in human disease.

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Centrin-Based Contractility

Centrins are small (20-25 kDa) acidic Ca2+-binding proteins that are highly conserved in all eukaryotic phyla. In addition to their conserved role in the duplication of centrioles, basal bodies, and spindle pole bodies, centrins are major constituents of contractile organelles in unicellular organisms. In Paramecium, for example, the infraciliary lattice (ICL) is a conspicuous cytoskeletal network underlying the whole cell surface and plays an important role in cellular Ca2+ homeostasis (Sehring et al. 2009). Although dispensable under laboratory conditions, its assembly is constitutively controlled by the basal bodies (Beisson et al. 2001). In response to a Ca2+ influx, contraction of the ICL drives cell contraction. The ICL is composed of a variety of centrins and high molecular mass components (Garreau de Loubresse et al. 1991; Madeddu et al. 1996; Klotz et al. 1997) that mediate Ca++-based contractility (Gogendeau et al. 2007). Analysis of the ICL also revealed a large expansion of centrin genes in P. tetraurelia. Two of the most conserved centrin subfamilies are physically and functionally restricted to basal bodies, whereas 10 more divergent subfamilies comprise ICL-specific proteins whose precise localization and function remain to be explored (Gogendeau et al. 2008). Tubulin Post-translational Modifications

Tubulin post-translational modifications (PTMs) occur widely in cilia and flagella. The molecular heterogeneity of tubulins (Bré et al. 1994; Redeker et al. 1994) combined with the diversity of microtubular networks (Cohen and Beisson 1980; Fleury et al. 1995) make Paramecium a suitable model for studying PTMs. Purification and analysis of axonemal tubulin from Paramecium cilia (Fig. 5) using monoclonal antibodies and mass spectrometry led to the discovery of a new type of PTM, polyglycylation (Redeker et al. 1994). This consists of adding polyglycine polymers onto several glutamate residues within the carboxy-terminal tail domain of α- and β-tubulins. This PTM is not specific to protists, occurring also in the cilia and flagella of metazoan cells (Adoutte et al. 1985; Bré et al. 1996; Dossou et al. 2007). Surprisingly, in Paramecium, the whole pool of purified tubulin C-terminal peptides is glycylated (Redeker et al. 1994; Bré et al. 1998); this is not the case in other ciliates, such as Tetrahymena (Redeker et al. 2005). Therefore, Paramecium is a good model for investigating the significance of this complex PTM in a single cell. In Paramecium, ciliary tubulin is not the only target of glycylation; glycylated tubulin isoforms partition asymmetrically based on the lengths of the polyglycine chains: Cytoplasmic and axonemal tubulins are modified by 1-9, and up to 34, glycine units, respectively (Bré et al. 1998). In organelles such as basal bodies, polyglycine chain lengthening is a marker of maturation during cell morphogenesis (Iftode et al. 2000). In contrast, during metazoan spermiogenesis, it is the appearance of polyglycylation that serves as a marker of spermatid differentiation (Bré et al. 1996). Polyglycylases are members of the tubulin tyrosine ligase-like family (Rogowski et al. 2009; Wloga et al. 2009). A phylogenetic analysis is in progress to identify glycylase genes in Paramecium. In addition, a reverse enzymatic activity has directly been found (Bré et al. 1998). Different genetic tools are available in Paramecium to overexpress or deplete the enzymes involved in polyglycylation, and altering their activity in the Paramecium model would likely contribute to a greater understanding of the importance of this PTM in morphogenesis.

FIGURE 5. Immunolabeling of microtubules. (Left) Cilia are labeled with an antibody against polyglycylated tubulin. (Right) Cilia are labeled in red, intracytoplasmic microtubules in green, and the MAC in blue. (Biphotonic microscopy courtesy of A. Aubusson-Fleury.) (For color figure, see doi: 10.1101/pdb.emo140 online at www. cshprotocols.org.) www.cshprotocols.org

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Electrophysiology and Behavioral Genetics

Paramecium is a useful model for cellular neurobiological investigations. Its large size makes it more amenable to electrophysiological investigation than Tetrahymena. Indeed, Paramecium was the first cell to be penetrated with an intracellular electrode, and later advances in two-electrode current clamp (Eckert 1972), voltage clamp (Oertel et al. 1977), and patch clamp (Saimi et al. 1992) placed Paramecium electrophysiology on firm footing. Paramecium also has an excitable membrane: When stimulated, it generates a Ca2+-based action potential. The resultant entry of calcium ions into the cilia causes them to reverse the direction of their beating, driving the cell backward. This “avoiding reaction,” a direct behavioral indicator of excitation, is easily monitored. Using this reaction as the phenotype, a number of behavioral mutants of Paramecium have been isolated; these mutants are amenable to electrophysiological studies to ascertain the missing or altered currents (Kung and Saimi 1982) and to transgenic manipulation. The complete genome sequence of Paramecium revealed a surprisingly large number of genes that encode ion channels, especially K+ channels. Although some of the gene products responsible for these behavioral effects are unique to ciliates, others are common to all eukaryotes. Interspecies sequence comparisons, together with the ability to knock down or knock out gene functions, open new avenues for the investigation of ion channels, membrane bioelectrics, and the control of motility.

GENETICS P. aurelia species are particularly well suited for classical genetic studies. In both conjugation and autogamy, only one of the haploid products from MIC meiosis survives in each cell, and an additional mitosis then gives rise to two genetically identical gametic nuclei in each post-meiotic cell (Fig. 2). In autogamy, fusion of the two gametic nuclei within a single cell therefore makes the zygotic genome entirely homozygous. In conjugation, reciprocal exchange of one of the gametic nuclei between the mates, followed by karyogamy, makes the zygotic genomes of the two mates strictly identical, whatever the genotypes of the parental cells. Paramecium genetics take advantage of these processes to analyze Mendelian traits. Autogamy is used after mutagenesis to allow the expression of recessive mutations in sexual progeny, and crosses between mutant and wild-type lines are used to analyze genetic determinism: The presence of different phenotypes in the two genetically identical mates immediately reveals non-Mendelian characters (Fig. 3). Known cases of maternally (i.e., cytoplasmically) inherited phenotypes fall into four classes: structural inheritance of cortical patterns based on protein-protein interactions (for review, see Beisson 2007), mitochondrial inheritance (Adoutte and Beisson 1970), inheritance of endosymbiotic bacteria (Preer et al. 1974), and “macronuclear inheritance,” which results from the homology-dependent mechanisms ensuring that the developing zygotic MAC will reproduce the particular genome rearrangement patterns observed in the maternal MAC of each cell (Meyer and Chalker 2007; Duharcourt 2009).

GENOMIC AND ASSOCIATED RESOURCES Numerous mutant strains of P. tetraurelia are available, including strains with recessive Mendelian alleles that provide good markers for genetic crosses because they confer easily scored phenotypes. Some examples are the alleles ts111-1 and ts401-1 (which confers lethality at 35°C), and nd7-1 (which produces trichocysts incapable of discharging). It also is possible to clone genes identified by recessive mutations through functional complementation, using a sib-selection approach (Haynes et al. 1998) and an indexed genomic library (Keller and Cohen 2000). ParameciumDB (http://paramecium.cgm.cnrs-gif.fr/) is the Paramecium community model organism database. It contains nomenclature guidelines for Paramecium, and integrates the genome sequence and annotations with genetic data (expressed sequence tags [ESTs], stock collection, RNAi experiments) and genome-related data sets such as orthologs in protists, plants, fungi, and animals, as well as data from proteome and transcriptome studies. It also provides advanced search capabilities, a Genome Browser, a Wiki for protocols, tutorials and other information, and tools for retrieval and alignment of gene, mRNA and protein sequences. The Paramecium Genome Browser at the Genoscope French National Sequencing Center (http://www.genoscope.cns.fr/externe/ GenomeBrowser/Paramecium/) contains the evidence used for automated annotation of nearly 40,000 protein-coding genes, including ab initio gene predictions and sequence reads. High-density 50-nt microarrays (three sense and three antisense probes per gene model) have been designed with NimbleGen and the design, NimbleGen “2006-09-12_Paramecium,” is freely available. www.cshprotocols.org

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TECHNICAL APPROACHES Standard procedures for maintaining P. tetraurelia lines of known clonal age through weekly cycles of vegetative growth and autogamy and their expansion by mass culture are presented in Maintaining Clonal Paramecium tetraurelia Cell Lines of Controlled Age through Daily Reisolation (Beisson et al. 2010a) and Mass Culture of Paramecium tetraurelia (Beisson et al. 2010b), respectively. A technique for Silencing Specific Paramecium tetraurelia Genes by Feeding Double-Stranded RNA (Beisson et al. 2010c), first developed in Caenorhabditis elegans, is a powerful and easily implemented method for reverse genetics. The genome can also be manipulated by DNA Microinjection into the Macronucleus of Paramecium (Beisson et al. 2010d). Procedures are also available for Immunocytochemistry of Paramecium Cytoskeletal Structures (Beisson et al. 2010e).

ACKNOWLEDGMENTS The authors acknowledge the support of the Centre National de la Recherche Scientifique and the Agence Nationale pour la Recherche.

REFERENCES Adoutte A, Beisson J. 1970. Cytoplasmic inheritance of erythromycin resistant mutations in Paramecium aurelia. Mol Gen Genet 108: 70–77. Adoutte A, Garreau de Loubresse N, Beisson J. 1984. Proteolytic cleavage and maturation of the crystalline secretion products of Paramecium. J Mol Biol 180: 1065–1081. Adoutte A, Claisse M, Maunoury R, Beisson J. 1985. Tubulin evclution: Ciliate-specific epitopes are conserved in the ciliary tubulin of Metazoa. J Mol Evol 22: 220–229. Arnaiz O, Malinowska A, Klotz C, Sperling L, Dadlez M, Koll F, Cohen J. 2010. Cildb: A knowledgebase for centrosomes and cilia. Database (in press). Aury J-M, Jaillon O, Duret L, Noël B, Jubin C, Porcel BM, Ségurens B, Daubin V, Anthouard V, Aiach N, et al. 2006. Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature 444: 171–178. Baudry C, Malinsky S, Restituito M, Kapusta A, Rosa S, Meyer E, Bétermier M. 2009. PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia. Genes & Dev 23: 2478–2483. Beale GH, Preer JR Jr. 2008. Paramecium: Genetics and epigenetics. CRC Press, Boca Raton, FL. Beisson J. 2007. Preformed cell structure and cell heredity. In Proteinbased inheritance (ed. YO Chernoff), pp. 106–118. Landes Biosciences, Austin, TX. Beisson J, Lefort-Tran M, Pouphile M, Rossignol M, Satir B. 1976. Genetic analysis of membrane differentiation in Paramecium. Freeze-fracture study of the trichocyst cycle in wild-type and mutant strains. J Cell Biol 69: 126–143. Beisson J, Clérot J-C, Fleury-Aubusson A, Garreau de Loubresse N, Ruiz F, Klotz C. 2001. Basal body-associated nucleation center for the centrin-based cortical cytoskeletal network in Paramecium. Protist 152: 339–354. Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L, Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010a. Maintaining clonal Paramecium tetraurelia cell lines of controlled age through daily reisolation. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5361. Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L, Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010b. Mass culture of Paramecium tetraurelia. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5362. Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L, Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010c. Silencing specific Paramecium tetraurelia genes by feeding double-stranded RNA. Cold Spring Harb Protoc (this issue). doi: 10.1101/ pdb.prot5363.

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Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L, Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010d. DNA microinjection into the macronucleus of Paramecium. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5364. Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L, Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010e. Immunocytochemistry of Paramecium cytoskeletal structures. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5365. Bétermier M. 2004. Large-scale genome remodelling by the developmentally programmed elimination of germ line sequences in the ciliate Paramecium. Res Microbiol 155: 399–408. Bonnemain H, Gulik-Krzywicki T, Grandchamp C, Cohen J. 1992. Interactions between genes involved in exocytotic membrane fusion in Paramecium. Genetics 130: 461–470. Bré M-H, de Néchaud B, Wolff A, Fleury A. 1994. Glutamylated tubulin probed in ciliates with the monoclonal antibody GT335. Cell Motil Cytoskeleton 27: 337–349. Bré M-H, Redeker V, Quibell M, Darmanaden-Delorme J, Bressac C, Cosson J, Huitorel P, Schmitter JM, Rossler J, Johnson T, et al. 1996. Axonemal tubulin polyglycylation probed with two monoclonal antibodies: Widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility. J Cell Sci 109: 727–738. Bré M-H, Redeker V, Vinh J, Rossier J, Levilliers N. 1998. Tubulin polyglycylation: Differential posttranslational modification of dynamic cytoplasmic and stable axonemal microtubules in Paramecium. Mol Biol Cell 9: 2655–2665. Caron F, Meyer E. 1985. Does Paramecium primaurelia use a different genetic code in its macronucleus? Nature 314: 185–188. Caron F, Meyer E. 1989. Molecular basis of surface antigen variation in Paramecia. Annu Rev Microbiol 43: 23–42. Catania F, Wurmser F, Potekhin AA, Przybo E, Lynch M. 2009. Genetic diversity in the Paramecium aurelia species complex. Mol Biol Evol 26: 421–431. Cohen J, Beisson J. 1980. Genetic analysis of the relationships between the cell surface and the nuclei in Paramecium tetraurelia. Genetics 95: 797–818. Dossou SJY, Bré M-H, Hallworth R. 2007. Mammalian cilia function is independent of the polymeric state of tubulin glycylation. Cell Motil Cytoskeleton 64: 847–855. Duharcourt S, Lepère G, Meyer E. 2009. Developmental genome rearrangements in ciliates: A natural genomic subtraction mediated by non-coding transcripts. Trends Genet. 25: 344–350. Duret L, Cohen J, Jubin C, Dessen P, Goût J-F, Mousset S, Aury J-M, Jaillon O, Noël B, Arnaiz O, et al. 2008. Analysis of sequence variability in the macronuclear DNA of Paramecium tetraurelia: A somatic view of the germline. Genome Res 18: 585–596.

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Eckert R. 1972. Bioelectric control of ciliary activity. Science 176: 473–481. Fleury A, Callen A-M, Bré M-H, Iftode F, Jeanmaire-Wolf R, Levilliers N, Clérot J-C. 1995. Where and when is microtubule diversity generated in Paramecium? Immunological properties of microtubular networks in the interphase and dividing cells. Protoplasma 189: 37–60. Forney JD, Blackburn EH. 1988. Developmentally controlled telomere addition in wild-type and mutant paramecia. Mol Cell Biol 8: 251–258. Froissard M, Keller A-M, Dedieu J-C, Cohen J. 2004. Novel secretory vesicle proteins essential for membrane fusion display extracellularmatrix domains. Traffic 5: 493–502. Garnier O, Serrano V, Duharcourt S, Meyer E. 2004. RNA-mediated programming of developmental genome rearrangements in Paramecium tetraurelia. Mol Cell Biol 24: 7370–7379. Garreau de Loubresse N, Klotz C, Viguès B, Rutin B, Beisson J. 1991. Ca2+-binding proteins and contractility of the infraciliary lattice in Paramecium. Biol Cell 71: 217–225. Gautier M-C, Garreau de Loubresse N, Madeddu L, Sperling L. 1994. Evidence for defects in membrane traffic in Paramecium secretory mutants unable to produce functional storage granules. J Cell Biol 124: 893–902. Gautier M-C, Sperling L, Madeddu L. 1996. Cloning and sequence analysis of genes coding for Paramecium secretory granule (trichocyst) proteins. A unique protein fold for a family of polypeptides with different primary structures. J Biol Chem 271: 10247–10255. Gogendeau D, Beisson J, Garreau de Loubresse N, Le Caer J-P, Ruiz F, Cohen J, Sperling L, Koll F, Klotz C. 2007. An Sfi1p-like centrinbinding protein mediates centrin-based Ca2+-dependent contractility in Paramecium tetraurelia. Eukaryot Cell 6: 1992–2000. Gogendeau D, Klotz C, Arnaiz O, Malinowska A, Dadlez M, Garreau de Loubresse N, Ruiz F, Koll F, Beisson J. 2008. Functional diversification of centrins and cell morphological complexity. J Cell Sci 121: 65–74. Görtz H-D. 2006. Symbiotic associations between ciliates and prokaryotes. In The prokaryotes: Vol. 1: Symbiotic associations, biotechnology, applied microbiology, 3rd ed (eds. M Dworkin et al.), pp. 364–402. Springer, New York. Haynes WJ, Vaillant B, Preston RR, Saimi Y, Kung C. 1998. The cloning by complementation of the pawn-A gene in Paramecium. Genetics 149: 947–957. Iftode F, Cohen J, Ruiz F, Torres Rueda A, Chen-Shan L, Adoutte A, Beisson J. 1989. Development of surface pattern during division in Paramecium. I. Mapping of duplication and reorganization of cortical cytoskeletal structures in the wild type. Development 105: 191–211. Iftode F, Clérot J-C, Levilliers N, Bré M-H. 2000. Tubulin polyglycylation: A morphogenetic marker in ciliates. Biol Cell 92: 615–628. Jaillon O, Bouhouche K, Goût J-F, Aury J-M, Noël B, Saudemont B, Nowacki M, Serrano V, Porcel BM, Ségurens B, et al. 2008. Translational control of intron splicing in eukaryotes. Nature 451: 359–362. Keller A-M, Cohen J. 2000. An indexed genomic library for Paramecium complementation cloning. J Eukaryot Microbiol 47: 1–6. Klotz C, Garreau de Loubresse N, Ruiz F, Beisson J. 1997. Genetic evidence for a role of centrin-associated proteins in the organization and dynamics of the infraciliary lattice in Paramecium. Cell Motil Cytoskeleton 38: 172–186. Kung C, Saimi Y. 1982. The physiological basis of taxes in Paramecium. Annu Rev Physiol 44: 519–534. Le Mouël A, Butler A, Caron F, Meyer E. 2003. Developmentally regulated chromosome fragmentation linked to imprecise elimination of repeated sequences in paramecia. Eukaryot Cell 2: 1076–1090. Lepère G, Bétermier M, Meyer E, Duharcourt S. 2008. Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia. Genes & Dev 22: 1501–1512.

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Lepère G, Nowacki M, Serrano V, Goût J-F, Guglielmi G, Duharcourt S, Meyer E. 2009. Silencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia. Nucleic Acids Res 37: 903–915. Madeddu L, Klotz C, Le Caer J-P, Beisson J. 1996. Characterization of centrin genes in Paramecium. Eur J Biochem 238: 121–128. Meyer E, Chalker DL. 2007. Epigenetics of ciliates. In Epigenetics (eds. CD Allis et al.), pp. 127–150. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Nowacki M, Zagorski-Ostoja W, Meyer E. 2005. Nowa1p and Nowa2p: Novel putative RNA binding proteins involved in transnuclear crosstalk in Paramecium tetraurelia. Curr Biol 15: 1616–1628. Oertel D, Schein SJ, Kung C. 1977. Separation of membrane currents using a Paramecium mutant. Nature 268: 120–124. Preer JR Jr, Preer LB, Jurand A. 1974. Kappa and other endosymbionts in Paramecium aurelia. Bacteriol Rev 38: 113–163. Preer JR Jr, Preer LB, Rudman BM, Barnett AJ. 1985. Deviation from the universal code shown by the gene for surface protein 51A in Paramecium. Nature 314: 188–190. Preer LB, Hamilton G, Preer JR Jr. 1992. Micronuclear DNA from Paramecium tetraurelia: Serotype 51A gene has internally eliminated sequences. J Protozool 39: 678–682. Redeker V, Levilliers N, Schmitter JM, Le Caer J-P, Rossier J, Adoutte A, Bré M-H. 1994. Polyglycylation of tubulin: A posttranslational modification in axonemal microtubules. Science 266: 1688–1691. Redeker V, Levilliers N, Vinolo E, Rossier J, Jaillard D, Burnette D, Gaertig J, Bré M-H. 2005. Mutations of tubulin glycylation sites reveal cross-talk between the C termini of α- and β-tubulin and affect the ciliary matrix in Tetrahymena. J Biol Chem 280: 596–606. Rogowski K, Juge F, van Dijk J, Wloga D, Strub JM, Levilliers N, Thomas D, Bré MH, Van Dorssaelaer A, Gaertig J, et al. 2009. Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation. Cell 137: 1076–1087. Saimi Y, Martinac B, Delcour AH, Minorsky PV, Gustin MC, Culbertson MR, Adler J, Kung C. 1992. Patch clamp studies of microbial ion channels. Methods Enzymol 207: 681–691. Scannell DR, Byrne KP, Gordon JL, Wong S, Wolfe KH. 2006. Multiple rounds of speciation associated with reciprocal gene loss in polyploid yeasts. Nature 440: 341–345. Sehring IM, Mansfeld J, Reiner C, Wagner E, Plattner H, Kissmehl R. 2007. The actin multigene family of Paramecium tetraurelia. BMC Genomics 8: 82. doi: 10.1186/1471-2164-8-82. Sehring IM, Klotz C, Beisson J, Plattner H. 2009. Rapid downregulation of the Ca2+-signal after exocytosis stimulation in Paramecium cells: Essential role of a centrin-rich filamentous cortical network, the infraciliary lattice. Cell Calcium 45: 89–97. Sonneborn TM. 1937. Sex, sex inheritance and sex determination in Paramecium aurelia. Proc Natl Acad Sci 23: 378–385. Sonneborn TM. 1950. Methods in the general biology and genetics of Paramecium aurelia. J Exp Zool 113: 87–147. Sonneborn TM. 1974. Paramecium aurelia. In Handbook of genetics (ed. RC King), pp. 469–594. Springer, New York. Strüder-Kypke MC, Wright A-DG, Fokin SI, Lynn DH. 2000. Phylogenetic relationships of the genus Paramecium inferred from small subunit rRNA gene sequences. Mol Phylogenet Evol 14: 122–130. Takahashi M, Naitoh Y. 1978. Behavioural mutants of Paramecium caudatum with defective membranes electrogenesis. Nature 271: 656–659. Wloga D, Webster D, Rogowski K, Bré MH, Levilliers N, JerkaDziadosz M, Janke C, Dougan ST, Gaertig J. 2009. TTLL3 is a tubulin glycine ligase that regulates the assembly of cilia. Dev Cell 16: 867–876. Zagulski M, Nowak JK, Le Mouël A, Nowacki M, Migdalski A, Gromadka R, Noël B, Blanc I, Dessen P, Wincker P, et al. 2004. High coding density on the largest Paramecium tetraurelia somatic chromosome. Curr Biol 14: 1397–1404.

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Protocol

Maintaining Clonal Paramecium tetraurelia Cell Lines of Controlled Age through Daily Reisolation Janine Beisson,1 Mireille Bétermier,1 Marie-Hélène Bré,2 Jean Cohen,1 Sandra Duharcourt,3 Laurent Duret,4 Ching Kung,5 Sophie Malinsky,3 Eric Meyer,3,7 John R. Preer Jr,6 and Linda Sperling1 1

Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, FRE3144, F-91198 Gif-sur-Yvette, France Laboratoire de Biologie Cellulaire 4, Centre National de la Recherche Scientifique, UMR 8080, Université Paris-Sud, 91405 Orsay Cedex, France 3 Laboratoire de Génétique Moléculaire, Centre National de la Recherche Scientifique, UMR 8541, École Normale Supérieure, F-75230 Paris, France 4 Laboratoire de Biométrie et Biologie Évolutive, Centre National de la Recherche Scientifique, UMR 5558, Université Lyon 1, F-69622, Villeurbanne, France 5 Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin-Madison, WI 53706, USA 6 Department of Biology, Indiana University, Bloomington, IN 47405-3700, USA 2

INTRODUCTION The sexual cycle of Paramecium tetraurelia can be managed by controlling food uptake, allowing the study of developmentally regulated differentiation in synchronous cultures. Clonal cell lines can be established easily by single cell isolation and can be grown in bacterized grass infusion medium for a large number of vegetative divisions before entering senescence. Sexual reproduction is triggered by starvation and therefore can be induced or prevented as needed by controlling the availability of food. Given a constant supply of food, P. tetraurelia cells will remain in the vegetative phase of their life cycle and, at 27°C, will divide by binary fission every 6 h. The daily reisolation procedure described here results in populations of P. tetraurelia of known age and produced under reproducible physiological conditions, which can help to standardize experiments. Paramecia produced under these conditions are suitable for immunocytochemical studies or use in genetic cross experiments.

RELATED INFORMATION The protocol described here (illustrated in Fig. 1) was established by Sonneborn (1950). For related protocols and more details, see http://paramecium.cgm.cnrs-gif.fr/parawiki/Protocols. Additional procedures are available for the Mass Culture of Paramecium tetraurelia (Beisson et al. 2010a). For additional information on the background, husbandry, and potential uses of Paramecium as model organisms, see Paramecium tetraurelia: The Renaissance of an Early Unicellular Model (Beisson et al. 2010b). Additional information may also be found in the ParameciumDB wiki: http:// paramecium.cgm.cnrs-gif.fr/parawiki/Protocols.

MATERIALS CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with , and

recipes for reagents marked with .

7

Corresponding author ([email protected]). Cite as: Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5361

© 2010 Cold Spring Harbor Laboratory Press

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Reagents Autogamy stain (optional; see Step 9.i)

DAPI (250 mg/mL) (optional; see Step 9.iv) Paramecium culture medium (standard)

Paramecium tetraurelia of the strain of interest

Equipment Sterilize all glassware before use. Note that although sterilized equipment is used, cells are not usually manipulated in a sterile environment.

Chamber, humidified Prepare this using a Pyrex baking dish with a Pyrex crystallizing dish inverted as a cover. Alternatively, a large plastic box with a tight-fitting lid can be used.

Glass plates, square or hexagonal The plates should fit easily in the humidified chamber.

Incubator preset to 27°C-28°C Micropipettes, glass Prepare by drawing out cotton-plugged Pasteur pipettes or glass tubing under a flame to a tip diameter of 100-200 µm.

Microscope, dissection, binocular Microscope, fluorescence, equipped with a filter set for monitoring green fluorescent protein (GFP) (if using DAPI; see Step 9.v) Microscope, phase-contrast (if using autogamy stain; see Step 9.ii) Microscope slides, glass Pipette bulb Slides, Pyrex, nine-well, 22-mm OD, 7-mm deep (Corning 7220-85) Three-well borosilicate slides (Fig. 2) are preferred because they are easier to manipulate, but must be custom ordered. It is convenient to have the front and back of each slide rough-ground to allow labeling with a charcoal pencil. Tissue culture plates (12- or 24-well) also can be used, although they are not as easy to manipulate and do not have very good optical properties for observing the cells.

Tubes, 20-mL Water bath (boiling, used to clean the micropipette between cell transfers)

FIGURE 1. Schematic diagram of the weekly reisolation protocol. www.cshprotocols.org

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FIGURE 2. Three-well slide.

METHOD Begin each cycle on a specific day (Monday is used as the first day in this protocol; see Fig. 1). 1. Working under a dissection microscope, isolate cells from the stock of each cell line of interest. Use

a micropipette to transfer a single cell to each of the wells of a glass slide, each containing ~200300 µL of medium. When transferring cells, always rinse the micropipette in boiling water after the transfer to avoid contamination between strains. The boiling water will kill any paramecia remaining in the micropipette. 2. Stack the slides in a humidified chamber, separated by glass plates (Fig. 3). Incubate overnight at

27°C-28°C. 3. The following day, count the cells in each well to evaluate the growth rate (i.e., number of

divisions). Cells of a wild-type reference strain should undergo four to five divisions per day under these conditions, except for the first day after autogamy (approximately three divisions). See Troubleshooting. 4. Replicate each clone by transferring a single cell to a well containing ~200-300 µL of medium in a

new slide. See Troubleshooting. 5. To the initial slide, add ~600-700 µL of medium to each well after the single cell has been

transferred. This allows each culture to reach a plateau of ~1000 cells per well. 6. Each day, check the clones for the characteristics of interest, e.g., mating type, thermosensitivity

at 35°C, or any other phenotypic trait corresponding to the genotype of the strain. 7. Each day, repeat Steps 4 and 5. See Troubleshooting. 8. On Friday, transfer the replicates to wells containing 1 mL of medium. Grow over the weekend

without reisolation. See Troubleshooting. 9. The following Monday, check each well for autogamy by one of the methods below. Examine at

least 50 cells per sample. Autogamous cells are recognized by a fragmented macronucleus (MAC) and the presence of two “anlage,” i.e., new, developing MAC (Fig. 4). See the Discussion and Paramecium tetraurelia: The Renaissance of an Early Unicellular Model (Beisson et al. 2010b) for details on the reproductive cycles of Paramecium.

FIGURE 3. Humidified chambers for Paramecium growth are assembled from a Pyrex baking dish and a Pyrex crystallizing dish inverted as a cover. Layers of welled slides are stacked within the chamber, separated by glass plates. A micropipette, used for cell transfer under a dissection microscope, is also shown. www.cshprotocols.org

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FIGURE 4. Fluorescent images of DAPI-stained cells. (A) Vegetative cell. Vegetative cells are easily recognized by the large MAC (yellow arrowhead). The two MIC can be seen under high magnification (red arrowheads). (B) Postautogamous cell. Fragmentation of the parental MAC indicates that the cell has recently gone through sexual reproduction. At late stages, the two developing new MAC (orange arrowheads) are large enough to be seen under low magnification. Magnification, 1000X. (For color figure, see doi: 10.1101/pdb.prot5361 online at www. cshprotocols.org.)

Using autogamy stain i.

Transfer a small drop (containing ~50 cells) from each well to a small drop of autogamy stain on a glass slide.

ii. Observe under a light microscope using phase-contrast optics. The MAC should be reddish and the cytoplasm green. See Troubleshooting.

Using DAPI iii. Transfer a small drop (containing ~50 cells) from each well to a glass slide. iv. Add a drop of DAPI. v. Observe under a fluorescence microscope. 10. Use individual cells from 100% autogamous cultures (as determined in Step 9) to start a new cycle. 11. Transfer the rest of each culture from the well to a 20-mL test tube containing 4 mL of fresh

medium. 12. Incubate for 24-48 h. These cultures can be stored for several weeks at 13°C-15°C and used for genetic crosses or to start a mass culture; for details, see Mass Culture of Paramecium tetraurelia (Beisson et al. 2010a).

TROUBLESHOOTING Problem: Medium becomes contaminated. [Step 3] Solution: Most problems encountered when growing paramecia can be attributed to contamination

of the medium. Prepare new micropipettes every day before reisolation of the cell lines. Problem: Cells grow poorly during daily isolation (e.g., fewer than three to four divisions for the wild

type per day) or become difficult to transfer because of formation of biofilms or the presence of sticky, filamentous bacteria. [Steps 4, 7, and 8] Solution: Clean cells by filling the depressions of a slide completely with Dryl’s buffer (for details on Dryl’s buffer, see Mass Culture of Paramecium tetraurelia [Beisson et al. 2010a]). Place a paramecium

at one end of the first well, let it swim to the other side, transfer the paramecium to the second well, let it swim to the other side, transfer it to the third well, etc. Return the bathed cell to fresh growth medium in a clean depression slide. Problem: The autogamy stain is too concentrated. [Step 9.ii] Solution: Dilute the stain with a mixture of equal proportions of ethanol, acetic acid, HCl, and H2O.

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DISCUSSION Given a constant supply of food, P. tetraurelia cells will remain in the vegetative phase of the life cycle and divide by binary fission every 6 h (at the standard temperature of 27°C). Under mild starvation conditions, cells become sexually reactive and will conjugate if they meet cells of a complementary mating type; if not, they will undergo autogamy (i.e., self-fertilization). Although starvation is required for induction of both autogamy and conjugation, autogamy cannot proceed unless cells have gone through a certain number of cell divisions since the previous sexual event (autogamy or conjugation). Some cells can undergo autogamy after 10-15 divisions, but 100% will go into autogamy after 20-25 cell divisions. In contrast, there is no immaturity period for conjugation in P. tetraurelia. In both conjugation and autogamy, the first cytologically detectable event is meiosis of the micronucleus (MIC), followed by fragmentation of the MAC into ~30 pieces, which remain transcriptionally active. Karyogamy of gametic nuclei results in a diploid zygotic nucleus, which then divides twice: Two of the products will become the new MIC of the sexual progeny, whereas the other two begin development into new MAC. At the first cellular division after sexual events (which requires refeeding), the two new MIC divide by mitosis, whereas the two new MAC (not yet fully mature) segregate without division to the two daughter cells. These daughter cells, called karyonides, start dividing by a nonmitotic process at the second cellular division. Fragments of the parental MAC stop replicating DNA but are not actively degraded if cells are refed within a few days of meiosis; they are simply randomly distributed to daughter cells at each cellular division (remaining active in transcription) until none are left.

REFERENCES Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L, Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010a. Mass culture of Paramecium tetraurelia. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5362. Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L,

www.cshprotocols.org

Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010b. Paramecium tetraurelia: The renaissance of an early unicellular model. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.emo140. Sonneborn TM. 1950. Methods in the general biology and genetics of Paramecium aurelia. J Exp Zool 113: 87–147.

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Cold Spring Harbor Protocols

Protocol

Mass Culture of Paramecium tetraurelia Janine Beisson,1 Mireille Bétermier,1 Marie-Hélène Bré,2 Jean Cohen,1 Sandra Duharcourt,3 Laurent Duret,4 Ching Kung,5 Sophie Malinsky,3 Eric Meyer,3,7 John R. Preer Jr,6 and Linda Sperling1 1

Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, FRE3144, F-91198 Gif-sur-Yvette, France Laboratoire de Biologie Cellulaire 4, Centre National de la Recherche Scientifique, UMR 8080, Université Paris-Sud, 91405 Orsay Cedex, France 3 Laboratoire de Génétique Moléculaire, Centre National de la Recherche Scientifique, UMR 8541, École Normale Supérieure, F-75230 Paris, France 4 Laboratoire de Biométrie et Biologie Évolutive, Centre National de la Recherche Scientifique, UMR 5558, Université Lyon 1, F-69622, Villeurbanne, France 5 Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin-Madison, WI 53706, USA 6 Department of Biology, Indiana University, Bloomington, IN 47405-3700, USA 2

INTRODUCTION Clonal cell lines of Paramecium tetraurelia can be established easily by single cell isolation. Given a constant supply of food, P. tetraurelia cells will remain in the vegetative phase of their life cycle and, at 27°C, will divide by binary fission every 6 h. P. tetraurelia is suitable for a range of biochemical and molecular studies such as mitochondrial genetics, post-translational tubulin modifications, variant genetic codes, programmed genome rearrangements (and their epigenetic regulation by noncoding RNAs), and RNA interference. This protocol describes the methods required to grow high-density mass cultures of P. tetraurelia in quantities sufficient to provide the material required for biochemical and molecular biological studies.

RELATED INFORMATION The protocol described here was established by Sonneborn (1950). For related protocols and more details, see http://paramecium.cgm.cnrs-gif.fr/parawiki/Protocols. Additional procedures are available for Maintaining Clonal Paramecium tetraurelia Cell Lines of Controlled Age through Daily Reisolation (Beisson et al. 2010a). For additional information on the background, husbandry, and potential uses of Paramecium as model organisms, see Paramecium tetraurelia: The Renaissance of an Early Unicellular Model (Beisson et al. 2010b). Additional information may also be found in the ParameciumDB wiki: http://paramecium.cgm.cnrs-gif.fr/parawiki/Protocols.

MATERIALS CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with , and

recipes for reagents marked with .

Reagents Dryl’s buffer

7

Corresponding author ([email protected]). Cite as: Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5362

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Paramecium culture medium (rich) Paramecium tetraurelia Prepare populations of the strain of interest as described in Maintaining Clonal Paramecium tetraurelia Cell Lines of Controlled Age through Daily Reisolation (Beisson et al. 2010a).

Equipment Bottles, pear-shaped Centrifuge, clinical (optional; see Step 9) Centrifuge, oil-testing Flasks, glass, Erlenmeyer or round-bottom Funnel, plastic or glass Incubator preset to 27°C Gauze, sterile Micropipettes, glass Prepare by drawing out cotton-plugged Pasteur pipettes or glass tubing under a flame to a tip diameter of 100-200 µm.

Microscope, dissection, binocular Pasteur pipettes Rod, glass, solid Tubes, glass or polycarbonate, 15- or 50-mL

METHOD Mass Culture In rich medium, it is possible to grow mass cultures of wild-type cells to a density of 3-6 × 103 cells/mL. 1. Fill Erlenmeyer or round-bottomed flasks approximately halfway with rich culture medium. The flasks must not be more than half full, to assure adequate oxygenation. 2. Inoculate cultures at a density of 40-100 cells/mL. Beneath this value, the culture could have trouble starting to grow or not grow at all. Above this value, the stationary phase might be reached more rapidly than intended. 3. Grow mass cultures without agitation at 27°C. The length of time required to reach the stationary phase will depend on the number of cells in the inoculum, the final volume, and the richness of the medium used. Generally, the length of incubation can be estimated based on a growth rate of ~four divisions per 24 h for the wild-type strain. 4. Evaluate the density of the culture: i.

Mix the culture by swirling gently.

ii. Remove three 1-mL aliquots. iii. Count the number of cells in each aliquot by capturing each cell individually with a glass

micropipette. In this way, if desired, accurate growth curves can be obtained. A culture has reached the stationary phase when it is no longer turbid, i.e., most bacteria have been eaten. See Troubleshooting.

Cell Harvesting Harvested cells can be used for biochemical analyses or inoculated into a larger volume for additional growth. 5. Remove grass debris and other flocculent material by filtering the culture through a tight plug of

several layers of sterile gauze in a funnel.

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6. Distribute the filtered cells into pear-shaped bottles. Collect the cells by centrifugation in an oil-

testing centrifuge at 170g-200g for 2 min. 7. Empty the bottles by decanting the liquid while the paramecia are trapped at the narrow end by

a glass rod (Fig. 1). Alternatively, remove the cells from the bottom of the bottle using a Pasteur pipette. 8. For use in biochemical studies, resuspend the pellet in 100 mL of Dryl’s buffer using a Pasteur pipette. Cells can be left for several hours in the Dryl’s buffer to purge them completely of the bacteria in the digestive vacuoles. 9. Centrifuge the cells at 170g-200g for 2 min. Discard the supernatant. 10. Repeat Steps 8 and 9 at least two to three times. In general, cultures should be prepared fresh for each experiment. Although cultures can be stored for a few days at 13°C (or up to 18°C), the lipid composition of the membranes changes; depending on the study, this might not be advisable. Long-term storage is not advised.

TROUBLESHOOTING Problem: A biofilm forms in the culture. [Step 4] Solution: Consider the following: 1. For mass cultures, it is advisable to filter the cultures through several layers of gauze packed tightly

into a plastic or glass funnel during expansion (i.e., when transferring a 50-mL preculture to 500 mL or more of fresh medium). This is critically important if any visible biofilm has formed. 2. It may be helpful to centrifuge and wash the cells in Dryl’s buffer before transfer to fresh medium.

FIGURE 1. Collecting Paramecium by centrifugation in pearshaped bottles. The glass rod is used to trap the cells in the narrow part of the bottle while decanting the liquid.

REFERENCES Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L, Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010a. Maintaining clonal Paramecium tetraurelia cell lines of controlled age through daily reisolation. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5361. Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L,

www.cshprotocols.org

Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010b. Paramecium tetraurelia: The renaissance of an early unicellular model. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.emo140. Sonneborn TM. 1950. Methods in the general biology and genetics of Paramecium aurelia. J Exp Zool 113: 87–147.

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Cold Spring Harbor Protocols

Protocol

Silencing Specific Paramecium tetraurelia Genes by Feeding Double-Stranded RNA Janine Beisson,1 Mireille Bétermier,1 Marie-Hélène Bré,2 Jean Cohen,1 Sandra Duharcourt,3 Laurent Duret,4 Ching Kung,5 Sophie Malinsky,3 Eric Meyer,3,7 John R. Preer Jr,6 and Linda Sperling1 1

Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, FRE3144, F-91198 Gif-sur-Yvette, France Laboratoire de Biologie Cellulaire 4, Centre National de la Recherche Scientifique, UMR 8080, Université Paris-Sud, 91405 Orsay Cedex, France 3 Laboratoire de Génétique Moléculaire, Centre National de la Recherche Scientifique, UMR 8541, École Normale Supérieure, F-75230 Paris, France 4 Laboratoire de Biométrie et Biologie Évolutive, Centre National de la Recherche Scientifique, UMR 5558, Université Lyon 1, F-69622, Villeurbanne, France 5 Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin-Madison, WI 53706, USA 6 Department of Biology, Indiana University, Bloomington, IN 47405-3700, USA 2

INTRODUCTION This protocol describes an easy and efficient method to knock down gene expression in a homologydependent manner by feeding Paramecium with bacteria-producing double-stranded (ds) RNA corresponding to a portion of the target gene. The target sequence is cloned in a plasmid between two convergent T7 promoters (the L4440 plasmid) and used to transform an E. coli strain devoid of RNase III activity and expressing the T7 polymerase gene under the control of an inducible Plac promoter (strain HT115-DE3). Inserts corresponding to ~400 bp of the coding sequence to be silenced are recommended; very short inserts (.

Reagents

β-sitosterol (4 mg/mL, prepared in 100% ethanol) Ampicillin, filter-sterilized (100 mg/mL) Dryl’s buffer (for large-scale cultures only)

Escherichia coli, HT115, carrying L4440 plasmids with relevant inserts targeting the Paramecium genes to be silenced Maintain on tetracycline to select for the mutated RNase III gene, which is inactivated by insertion of a transposon carrying a tetracycline resistance gene. Plasmid L4440 confers resistance to ampicillin.

IPTG stock solution (0.8 M) LBAT LBAT-agar plates Paramecium of the strain of interest Wheat grass medium (standard or rich) Use standard medium for small-scale cultures (e.g., for phenotypic observation) and rich medium for mass cultures for biochemical or molecular analyses.

Equipment Bottles, pear-shaped (for large-scale cultures only) Centrifuge, clinical Centrifuge, oil-testing (for large-scale cultures only) Flasks, glass, Erlenmeyer or round-bottom (for large-scale cultures only) Incubator preset to 27°C Incubator, shaking, preset to 37°C Inoculation loop Micropipettes, glass Prepare by drawing out cotton-plugged Pasteur pipettes or glass tubing under a flame to a tip diameter of 100-200 µm.

Microscope, dissection Pasteur pipettes Rod, glass, solid (for large-scale cultures only) Slides, Pyrex, nine-well, 22-mm OD, 7-mm deep (Corning 7220-85) Three-well borosilicate slides are preferred because they are easier to manipulate, but must be custom ordered. It is convenient to have the front and back of each slide rough-ground to allow labeling with a charcoal pencil. Tissue culture plates (12- or 24-well) also can be used, although they are not as easy to manipulate and do not have very good optical properties for observing the cells.

Spectrophotometer

METHOD Procedures are described for preparing silencing medium in either LB (Steps 1-7) or directly into wheat grass medium (Steps 8-12), as well as for small-scale cultures for morphological and/or behavioral observations (Step 13) and largescale cultures to generate sufficient source material for biochemical and molecular studies (Step 14).

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Preparation of Silencing Medium Using LB 1. Start a bacterial preculture by inoculating 10 mL of LBAT with a single E. coli HT115 colony picked

from an LBAT-agar plate. Incubate with shaking overnight at 37°C. 2. The following day, dilute the overnight preculture 100-fold in LBAT. Grow with shaking at 37°C to

an OD600 of 0.4-0.6. 3. Add IPTG to a final concentration of 0.4 mM. Continue shaking for 3-4 h at 37°C. 4. Centrifuge the IPTG-induced culture at 3000g for 10 min. 5. Remove the supernatant. Wash the pellet twice with an equivalent volume of wheat grass medium

(i.e., unbacterized Paramecium culture medium). 6. Resuspend the pellet in wheat grass medium containing 0.4 mM IPTG and 100 µg/mL ampicillin,

adjusting the final OD to 0.1-1.0 as desired. With an OD of 0.1, the culture should reach a density of ~1000 cells/mL before starving. Do not add tetracycline; Paramecium does not tolerate it very well.

7. Add 1 µL of β-sitosterol per each 5 mL of medium.

The silencing medium will remain biologically active for ~24 h, provided it is not contaminated with some other ampicillin-resistant bacterium, which can outgrow the IPTG-induced HT115 E. coli strain. To silence a gene continuously during several days of vegetative growth, prepare fresh medium every day.

Preparation of Silencing Medium Using Wheat Grass Medium It is often simpler to grow and induce the HT115 strain directly in wheat grass medium. This is the method of choice for silencing genes that are expressed specifically during sexual reproduction or early development, because the lower bacterial density allows an easier control of the timing of meiosis, which is triggered by starvation when cells have eaten all of the bacteria. 8. Start a bacterial preculture by inoculating 10 mL of LBAT with a single E. coli HT115 colony picked

from an LBAT-agar plate. Incubate with shaking overnight at 37°C. 9. The following day, dilute the overnight preculture 100-fold in wheat grass medium (i.e., unbacterized

Paramecium culture medium) containing 100 µg/mL ampicillin. Grow overnight with shaking at 37°C. Shaking at 37°C allows E. coli to grow almost to the same density as K. pneumoniae in unshaken culture medium at 27°C (i.e., to an OD of ~0.05-0.07 in standard medium, or ~0.15 in rich medium). 10. The following day, dilute the saturated E. coli culture four- to sixfold in wheat grass medium con-

taining 100 µg/mL ampicillin. Shake for 30 min at 37°C. 11. Add IPTG to a final concentration of 0.4 mM. Continue shaking for at least 4 h or overnight at 37°C. 12. Add 1 µL of β-sitosterol per each 5 mL of medium.

Small-Scale Experiments This method is useful if the expected phenotype can be observed with a small number of cells. 13. Process the cells as desired for the gene of interest:

If gene silencing does not impair vegetative growth i.

Using a glass micropipette, introduce a single paramecium into 200-300 µL of freshly prepared standard silencing medium (from Step 7 or Step 12) in the well of a slide. Incubate overnight at 27°C.

Three hundred microliters of medium with an OD ≥0.05 will provide sufficient food to maintain cells in exponential growth for 24 h, assuming a wild-type growth rate of four to five divisions per day at 27°C.

ii. Optionally, if appearance of the phenotype requires a larger number of divisions, isolate a

single cell the following day. Transfer to 200-300 µL of fresh standard silencing medium, using the daily reisolation procedure described in Maintaining Clonal Paramecium tetraurelia Cell Lines of Controlled Age through Daily Reisolation (Beisson et al. 2010a).

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If higher cell densities are desired, use rich silencing medium instead. In either case, to maintain silencing continuously over several days, plan medium preparation in advance, such that freshly induced medium is available every day.

If the silenced gene is not expressed during vegetative growth There is no need to allow any number of vegetative divisions in the silencing medium before meiosis, which is triggered by starvation when cells have eaten all of the bacteria. To silence genes that are expressed during sexual reproduction, take care to ensure that meiosis will occur CsCl As an alternative to CsCl gradients, DNA purification columns can be used (see Step 3).

DNA to be microinjected DNA, salmon sperm (optional; see Step 4) Dryl’s buffer containing 0.2% bovine serum albumin (Dryl-BSA) Ethanol Paraffin oil Paramecium of the strain of interest Paramecia react to aggression (such as microinjection) by firing secretory granules (trichocysts); this does not prevent microinjection but can make it more difficult. Some researchers circumvent this problem by using a mutant strain unable to fire trichocysts, e.g., nd7-1; co-injection of the wild-type ND7 gene with the construct of interest can be used to select successfully transformed clones by testing for trichocyst discharge. Alternatively, trichocysts can be removed just before microinjection by treatment with aminoethyl dextran. Large amounts of this compound can be synthesized easily (Plattner et al. 1984; Kerboeuf and Cohen 1990); small samples can be obtained by request from [email protected]. Paramecium culture medium (standard) Phenol Restriction enzymes (optional; see Step 1)

Equipment Centrifuge Alternatively, centrifugal devices such as Ultrafree-MC (Millipore) can be used before ethanol precipitation (see Step 3 note).

Coverslips, glass, 24- × 50-mm Microinjector, air-pressure-operated, equipped with tubing and microinjection needle holder Microinjector, manually operated, equipped with tubing and microinjection needle holder Micromanipulators Micropipettes, glass Prepare by drawing out cotton-plugged Pasteur pipettes or glass tubing under a flame to a tip diameter of 100-200 µm.

Microscope, inverted, equipped with phase-contrast optics and 10X and 20X/40X objectives Needles, microinjection, 1- and 10-µm-wide tips (e.g., Eppendorf femtotips) Pipettor, automatic, and tips (e.g., Eppendorf microloaders) Slides, Pyrex, nine-well, 22-mm OD, 7-mm deep (Corning 7220-85) Three-well borosilicate slides are preferred because they are easier to manipulate but must be custom ordered. It is convenient to have the front and back of each slide rough-ground to allow labeling with a charcoal pencil. Tissue culture plates (12- or 24-well) also can be used, although they are not as easy to manipulate and do not have very good optical properties for observing the cells.

METHOD Preparation of DNA 1. If the gene is cloned into a circular plasmid, linearize the plasmid by cutting at a unique restriction

site within the vector sequence.

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This avoids the random linearization that occurs in the macronucleus after injection of a circular molecule, before multimerization and telomere addition. This is particularly important for long genes, because most molecules would be cut within the coding sequence, producing high copy numbers that can silence intact copies and homologous endogenous genes post-transcriptionally. 2. After digestion, extract the DNA with phenol. Precipitate with ethanol. 3. Purify the DNA using CsCl gradients or purification columns; minipreparations are not appropriate. Even with highly purified DNA, microscopic dust particles are often present; a 1-h centrifugation of the DNA at 18,000g is recommended before microinjection. Alternatively, dilute DNA solutions can be filtered using centrifugal devices, such as Ultrafree-MC (Millipore) before ethanol precipitation. 4. Dissolve DNA in H2O to at least 5 mg/mL. The viscosity of such solutions allows better control of the amount delivered. If a lower concentration is needed, dilute DNA in salmon sperm DNA before precipitation and dissolution in H2O.

Preparation of Cells Paramecium cells should be from “young” clones (i.e., recipes for reagents marked with .

Reagents Antibody, primary, targeted against the epitope of interest For the rapid labeling method, use high-affinity monoclonal primary antibodies.

Antibody, secondary, targeted against the species in which the primary antibody was raised Bovine serum albumin (BSA; 3% in TBSTEM)

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Corresponding author ([email protected]). Cite as: Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5365

© 2010 Cold Spring Harbor Laboratory Press

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Vol. 5, Issue 1, January 2010

Aliquot into small tubes. Store at -20°C.

Citifluor mountant media (AF2; Citifluor Ltd.) DNA dye (e.g., Hoescht, DAPI) Nail polish (optional; see Step 17) Paraformaldehyde (2% [w/v], prepared in 1X PHEM) Aliquot into small tubes. Store at -20°C. Use within 2 d of thawing. PHEM (5X) TBSTEM Triton X-100 (1% [v/v], prepared in 1X PHEM) Aliquot into small tubes. Store at -20°C.

Equipment Centrifuge, clinical (optional; for labeling large cell populations only) Coverslips Micropipettes, glass Prepare by drawing out cotton-plugged Pasteur pipettes or glass tubing under a flame to a tip diameter of 100-200 µm.

Microscope, dissection, binocular Microscope, fluorescence, equipped with a filter set for monitoring green fluorescent protein Microscope slides, glass Pipette bulb Slides, Pyrex, nine-well, 22-mm OD, 7-mm deep (Corning 7220-85) Three-well borosilicate slides are easier to manipulate but must be custom ordered.

METHOD In addition to the standard method of antibody labeling of cells (Steps 1-9), a simplified protocol is described for rapidly screening many different clonal populations (Steps 10-15).

Antibody Labeling of Cells For all steps, handle cells individually under the dissection microscope in slide wells. Alternatively, large populations of cells can be labeled simultaneously using essentially the same procedure as that described below. However, instead of transferring individual cells, pellet the cells gently by centrifugation (e.g., ~200g-300g for 3 min in a clinical centrifuge) and resuspend in each successive medium. 1. Collect cells (30-50) in as small a volume of liquid as possible (~30-40 µL) in one well. 2. Add 300 µL of 1% Triton X-100 in PHEM. Incubate for 3-5 min at room temperature. For mass labeling of large populations, the resuspension volume for this permeabilization step is particularly crucial: Use ~5 mL of Triton in PHEM for a ~0.1-mL cell pellet. 3. Transfer cells to 2% paraformaldehyde in PHEM. Incubate for 10 min at room temperature. 4. Wash twice (~5-15 min each wash, at room temperature) by transferring cells in as little medium

as possible to wells containing 300 µL of 3% BSA in TBSTEM. The incubation time in each wash is not critical. 5. Transfer the cells to 50-100 µL of primary antibody in 3% BSA in TBSTEM. Incubate for 10-60 min

at room temperature. Determine the optimal antibody dilution empirically for each experiment. During incubations, cover the well with a microscope slide and place in a humidified chamber to prevent evaporation. 6. Transfer the cells to a well containing 300 µL of 3% BSA in TBSTEM. Incubate for ~5-15 min at

room temperature.

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7. Transfer cells to 50-100 µL of the secondary antibody in 3% BSA in TBSTEM. Incubate for 10-30

min at room temperature. As with the primary antibody, optimize the antibody dilution used for each experiment. 8. Transfer cells to a well containing a DNA dye (e.g., Hoescht, DAPI) in 3% BSA in TBSTEM. The concentration of dye used will depend on the objective of the experiment (i.e., whether strong or weak staining of the DNA is required). For example, 2 µL/mL of a 1 mg/mL Hoescht dye stock solution can be used to stain nuclei in experiments designed to study the cytoskeleton. 9. Proceed to Step 16.

Rapid Labeling of Individual Cells For immunostaining of Ca2+-binding structures, use Tris-buffered saline containing 1% Tween 20, but without the EGTA and Mg2+. 10. Collect cells (30-50) in as small a volume of liquid as possible (~30-40 µL) in one well. 11. Add 300 µL 1% Triton X-100 in PHEM. Incubate for 1-2 min at room temperature. 12. Add 300 µL 2% paraformaldehyde in PHEM to the well. Incubate for 10 min at room temperature. Add the fix directly to the well, rather than transferring the cells. 13. Wash once by transferring cells in as little medium as possible to wells containing 300 µL of TBSTEM. 14. Transfer the cells to 50-100 µL of a mixture of high-affinity monoclonal primary antibody and

secondary antibody in TBSTEM. Stir gently. 15. Incubate for 10 min at room temperature. Proceed to Step 16.

Mounting 16. Transfer cells in as little liquid as possible to a small (≤10 µL) drop of Citifluor. Cover gently with a

clean coverslip. To obtain optical sections using a confocal microscope, the cells should be mounted in a larger volume of Citifluor to avoid flattening. 17. If desired, seal the coverslip with nail polish once the liquid has spread uniformly.

DISCUSSION Although the buffers used in this protocol are well adapted to Paramecia, the incubation times listed for Triton, paraformaldehyde, and the antibodies can be adjusted according to the specificity of the antibody and the required level of denaturation of the antigens or the accessibility of the recognized epitopes. Certain antibodies could require longer fixation and/or incubation times. For example, the “universal” anti-α-tubulin DM1A must be fixed for up to 45 min to reveal all the microtubule networks. Most cytoskeletal structures are better preserved if the EGTA/Mg2+ is present in the permeabilization and fixation buffers and maintained throughout the staining procedure. In contrast, immunostaining of Ca2+-binding structures might preclude the use of EGTA/Mg2+, instead requiring TBST. Similarly, the permeabilization time (Triton) and fixation time (paraformaldehyde) can be modulated (1-15 and 10-60 min, respectively) as a function of the properties (solubility, accessibility, etc.) of the antigen of interest. For most polyclonal primary antibodies, a short incubation time (10 min, as indicated) is optimal to minimize nonspecific staining, which is often observed because of the thickness of the cell and its numerous basal bodies, and the presence of trichocysts that can trap antibodies nonspecifically.

REFERENCES Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L, Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010a. Maintaining clonal Paramecium tetraurelia cell lines of controlled age through daily reisolation. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5361.

www.cshprotocols.org

Beisson J, Bétermier M, Bré M-H, Cohen J, Duharcourt S, Duret L, Kung C, Malinsky S, Meyer E, Preer JR Jr, et al. 2010b. Paramecium tetraurelia: The renaissance of an early unicellular model. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.emo140.

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Appendix 1: Recipes [NOTE: This print edition of CSH Protocols contains only recipes for reagents requiring multiple components or nonobvious critical steps. Recipes for reagents marked with the symbol not listed below can be found online at http://www.cshprotocols.org/recipes.]

Acetocarmine MATERIALS Reagents Acetic acid (100%) Carmine powder (e.g., Sigma C1022)

Equipment Filter paper Water bath (boiling)

METHOD 1. 2. 3. 4.

Pour 55 mL of boiling water plus 45 mL of 100% acetic acid on 0.5 g of carmine powder. Mix. Place in a boiling water bath until the mixture starts to boil again. Mix again. Boil a few more minutes. Allow to cool. Filter.

Achromopeptidase solution Achromopeptidase (60 U/mL) NaCl (10 mM) Tris-Cl (10 mM, pH 8.0) Prepare fresh immediately before use.

AEC buffer AEC tablets (aminoethylcarbazole, 3-amino-9-ethylcarbazole; 20 mg) (Sigma) DMF (N,N-dimethylformamide) (Fisher Scientific) Acetic acid (0.1N) Sodium acetate (0.1M) Dissolve one AEC tablet (20 mg) in 2 mL of DMF. Add this solution to a final concentration of 0.03% to a 1:2.3 solution of 0.1N acetic acid and 0.1M sodium acetate. Prepare AEC solution within minutes of use.

AMEM (1%) 24 mL H2O (sterile; Merck) 1 mL antibiotic-antimycotic solution (AA solution; Sigma) 1 mL glutamine (Invitrogen) 1 mL insulin solution for amphibians 100 µL gentamicin (50 mg/mL; Sigma) 1 mL fetal calf serum, embryonic stem (ES) cell-qualified, heat-inactivated (Invitrogen) (see Note below) 72 mL MEM (1X; Invitrogen)

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Prepare the medium in a sterile reagent bottle (e.g., a 75-cm2 tissue culture flask). Combine the reagents in the order listed. The resulting culture medium is MEM-adjusted to amphibian osmolarity plus serum for growth. To heat-inactivate the complement system in the serum, defrost the stock bottle and incubate for 30 min at 56ºC. Cool the bottle to room temperature and store as 10-mL aliquots at –20ºC.

Amphibian dissection medium Reagent

Amount to add

Final concentration

APBS

5 mL 5 µL

50 µg/mL

50 mg/mL gentamicin (Sigma)

Ampicillin stock solution (100 mg/mL) Ampicillin (sodium salt [sodium ampicillin], m.w. = 371.40) Dissolve 1 g of sodium ampicillin in sufficient H2O to make a final volume of 10 mL. If sterilization is required, prewash a 0.45- or 0.22-µm sterile filter by drawing through 50-100 mL of H2O. Then pass the ampicillin solution through the washed filter. Store the ampicillin in aliquots at –20°C for 1 yr (or at 4°C for 3 mo).

APBS 25 mL H2O (sterile; Merck) 100 mL PBS (phosphate-buffered saline; Invitrogen) In this recipe, PBS is adjusted to amphibian osmolarity (225 ± 5 mOsm/L) by the addition of H2O.

Araldite (ready to polymerize) Amount to prepare (mL)

Araldite (CY212) (g)

Araldite hardener (H 964) (g)

Araldite enhancer (DY 964) (mL)

10 30 50 70 100 130 150 180 200 250

5.56 16.95 28.25 39.55 56.50 74.00 84.75 101.70 113.00 141.25

5.56 16.95 28.25 39.55 56.50 74.00 84.75 101.70 113.00 141.25

0.2 0.6 1.0 1.4 2.0 2.6 3.0 3.6 4.0 5.0

Mix the desired amounts of ingredients in a glass container according to the total volume needed. Store in 5-mL aliquots for up to 12 mo at –20°C.

Autogamy stain Reagent

Quantity (for 18 mL)

Acetic acid (45%) Acetocarmine Fast Green (1% [w/v], prepared in 100% ethanol) HCl (1 N)

4.5 mL 10.5 mL 1 mL 2 mL

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These proportions are recommended, but if one of the stains is too concentrated (the macronucleus should be reddish and the cytoplasm green when observed under a light microscope), add the other one and check again. Repeat until the desired result is obtained. This stain (Dippell RV. 1955. A temporary stain for Paramecium and other ciliate protozoa. Stain Technol 30: 69–71) can be kept at room temperature for years.

B2S B2 medium (Laboratoire CCD) Fetal bovine serum, heat-inactivated (10%)

Blocking buffer for ELISPOT assay 1X PBS (10X PBS [Roche] diluted to 1X with water) 10 g/L bovine serum albumin (BSA) (Sigma) Filter through a 22-µM filter (Millipore).

Blocking solution Blocking reagent (Roche, 11096176001) 10X MAB Dissolve the blocking reagent as a 10% (w/v) stock in 1X MAB by heating it in a microwave (heat twice for 30 sec at maximum power; adjust according to your microwave). Freeze aliquots of the stock solution at –20°C.

Blocking solution for Drosophila PBS (10X) without potassium salt, diluted to 1X

1% BSA (bovine serum albumin) 0.05% Triton X-100 If a milder detergent is necessary, use 0.1% saponin.

Blocking solution for rabbit DEPC-PBS (10X), diluted to 1X

Normal goat serum (NGS) (1%, v/v) Triton X-100 (0.1%, v/v) Complement-deactivate NGS by incubation for 1 h at 60°C. Store 2-mL aliquots of NGS for up to 12 mo at –20°C.

Ca2+ calibrating solution 130 mM NaCl 3 mM KCl 10 mM HEPES-KOH 10 mM glucose 0.01 mM ionomycin (pH 7.3) The desired Ca2+ concentration can be achieved by adding Ca2+/EGTA or Ca2+/BAPTA buffer. Appropriate concentrations of Ca2+ and EGTA/BAPTA can be calculated using widely available calculators (e.g., http://www.stanford. edu/~cpatton/webmaxcE.htm).

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CARD-FISH amplification buffer Reagent

Amount to add

Blocking solution Dextran sulfate NaCl (5 M) Phosphate-buffered saline (PBS; 10X) H2O

0.4 mL 2.0 g 16 mL 4.0 mL to 40 mL

Mix all reagents in a 50-mL tube. Heat to 40°C-60°C. Shake until the dextran sulfate dissolves completely. Aliquots of the buffer can be stored for several weeks at 4°C.

CARD-FISH hybridization buffer Reagent

Amount to add

Blocking solution Dextran sulfate H2O Formamide, molecular biology grade (70%) NaCl (5 M) SDS (sodium dodecyl sulfate; 20%, w/v) Tris-Cl (1 M, pH 8.0)

2.0 mL 2.0 g X mL (see below) (14 - X) mL (see below) 3.6 mL 20 µL 0.4 mL

Mix all reagents in a 50-mL tube. Heat to 40°C-60°C. Shake until the dextran sulfate dissolves completely. Aliquots of the buffer can be stored for several months at –20°C. The stringency of the buffer can be varied as desired: % formamide

mL formamide (14 - X)

mL water (X)

20 25 30 35 40 45 50 55 60 65 70

4 5 6 7 8 9 10 11 12 13 14

10 9 8 7 6 5 4 3 2 1 0

CARD-FISH proteinase buffer EDTA (0.05 M) Tris-Cl (0.1 M, pH 8.0)

Coating buffer 1X PBS (10X PBS [Roche] diluted to 1X with H2O) 3 µg/mL of IFN-γ capture monoclonal antibody (mAb) and/or 3 µg/mL of IL-2 capture mAb (both from Becton Dickinson/Pharmingen) The dual coating buffer contains both mAbs, whereas the IL-2 coating buffer contains only the IL-2 capture mAb and the IFN-γ coating buffer contains only the IFN-γ capture mAb. Wells coated with the latter two buffers are

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used to compensate for the color sensitivity of the CTL analyzer during spot counting. Typically, for wells coated with IL-2 or IFN-γ coating buffers, cells plated in the wells are stimulated with a positive control stimulus (CEF or anti-CD3) in order to obtain a signal suitable for calibration. Coating buffer should be prepared within a few hours of use.

DAB staining solution for Drosophila 0.5 mg/mL DAB (3,3′-diaminobenzidine tetrahydrochloride) 0.003% H2O2 1.5 mM CoCl2 1.5 mM NiCl2 The addition of CoCl2 and NiCl2 is optional. They intensify the reaction product and give it a blue-black color; in the absence of these compounds, the reaction product will be brown. Soak all materials that become contaminated with DAB overnight in bleach prior to disposal.

DAB substrate DAB (3,3′-diaminobenzidine tetrahydrochloride) (1 mg/mL; e.g., Sigma D5637) K/Na-phosphate buffer (67 mM, pH 7.4) Immediately before use, add 10 µL/mL of 3% H2O2 (hydrogen peroxide) (i.e., to a final H2O2 concentration of 0.03%).

DEPC-H2O Mix 1 mL of DEPC (diethyl pyrocarbonate) with 1000 mL of water in a screw-cap glass bottle. Incubate for ~2 h at room temperature in a fume hood with occasional swirling. Autoclave. Store at room temperature for up to 12 mo.

DEPC-PBS (10X) Reagent

Amount to add

Final concentration (10X)

KCl KH2PO4 Na2HPO4•2H2O NaCl DEPC-H2O

2.01 g 1.36 g 17.8 g 80.07 g to 1000 mL

27 mM 10 mM 100 mM 1.37 M —

Dissolve all reagents in 800 mL of DEPC-H2O. If necessary, adjust the pH to 7.0. Add DEPC-H2O to 1000 mL. Filter-sterilize. Store for up to 12 mo at room temperature.

Detection buffer for ELISPOT assay 1X PBS (10X PBS [Roche] diluted to 1X with H2O) 0.05% Tween 20 10 g/L bovine serum albumin (BSA) 0.5 µg/mL fluorescein isothiocyanate (FITC)-conjugated anti-IFN-γ monoclonal antibody (mAb) (Mabtech) 0.5 µg/mL biotin-conjugated anti-IL-2 mAb (Becton Dickinson/Pharmingen) Detection buffer should be prepared within a few hours of use.

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Developing buffer for ELISPOT assay 1X PBS (10X PBS [Roche] diluted to 1X with H2O) 0.05% Tween 20 10 g/L bovine serum albumin (BSA) 0.5 µg/mL mouse horseradish peroxidase (HRP)-conjugated anti-fluorescein isothiocyanate (FITC) IgG monoclonal antibody (mAb) (Cedarlane Laboratories) 0.5 µg/mL streptavidin alkaline phosphatase (Cedarlane Laboratories)

Dryl’s buffer Reagent

Quantity (for 4 L)

Final concentration

H2O Sodium citrate (0.1 M) NaH2PO4 (0.1 M) Na2HPO4 (0.1 M) CaCl2 (0.1 M) H2O

~3.6 L 80 mL 40 mL 40 mL 60 mL to 4 L

— 2 mM 1 mM 1 mM 1.5 mM —

Add reagents in the order listed. Add CaCl2 last (just prior to the second addition of H2O, which brings the volume to 4 L) to avoid precipitation of calcium phosphate. The solution was developed by Dryl (Dryl SJ. 1959. Antigenic transformation in Paramecium aurelia after treatment during autogamy and conjugation. J Protozool 6: S25).

EDTA To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA•2H2O to 800 mL of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (~20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to ~8.0 by the addition of NaOH.

Erythrosine B dye 1X PBS (10X PBS [Roche] diluted to 1X with H2O) 10% fetal bovine serum (FBS) (Wisent) 1.5 g/L Erythrosine B (Sigma)

Extracellular saline solution 135 mM NaCl 3 mM KCl 2 mM CaCl2 20 mM glucose 20 mM HEPES-NaOH (pH 7.4)

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Feeding bacteria MATERIALS Reagents LB-ampicillin agar plates Glycerol, sterile Klebsiella pneumoniae, wild-type, avirulent, ampicillin-resistant Although this is the standard feeding bacterial strain, other enterobacteria can be used. LB (Luria-Bertani) liquid medium

Equipment Freezer preset to –80°C Incubator preset to 37°C Inoculation loop Refrigerator preset to 4°C Tubes, microcentrifuge, 2-mL

METHOD 1. Grow the bacterial culture overnight in LB liquid medium. 2. Distribute 0.5-mL aliquots of the culture into 2-mL tubes containing 0.5 mL glycerol. Store tubes at –80°C. 3. Each week, thaw one tube. Streak thawed culture onto one (or more) LB-ampicillin plates. Incubate overnight at 37°C. 4. The following day, streak each of seven tubes with one colony from the Petri dish. Incubate overnight at 37°C. Store cultures at 4°C for use the following day.

Freezing diluent 88 mM citric acid (Sigma C2404) 47 mM D-(+)-glucose (Sigma G6152) 3.5 M DMSO (dimethyl sulfoxide; Sigma D2650) 0.1 M sucrose (Sigma S1888) 0.25 M Tris base (Trizma base; Sigma T6066)

Gelatin/BSA embedding medium Reagent

Amount to add (for 500 mL)

Final concentration (w/v)

Gelatin Bovine serum albumin (BSA) Sucrose DEPC-PBS (10X), diluted to 1X

2.2 g 135 g 90 g to 500 mL

0.4% 27% 18% —

Add the gelatin, BSA, and the sucrose to 450 mL of DEPC-PBS, stirring constantly until all ingredients are dissolved. Adjust the volume to 500 mL. Store 50-mL aliquots for up to 12 mo at –20°C. Store thawed aliquots refrigerated at 4°C; use within 1 wk.

HM199S Fetal bovine serum, heat-inactivated (10%) Medium 199, HEPES modification (Sigma M7528)

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Holding pipettes MATERIALS Equipment Capillary tubes, borosilicate glass, 10-cm length, 1-mm OD, 0.5-mm ID (e.g., Clark or equivalent) Container (clean, airtight) Ethanol burner

METHOD 1. Using an ethanol burner, gently heat the capillary tubes ~2 cm from one tip. 2. Bend the glass until the desired angle is obtained. The angle will vary according to the type of micromanipulator used. 3. Carefully flame-polish the capillary opening of the bent tip. Non-polished capillary openings might damage (cut into) the immobilized blastocyst. 4. Store holding pipettes in a clean airtight container until use.

Hybridization solution for rabbit Reagent

Amount to add (for 50 mL)

Final concentration

DEPC-H2O

18.6 mL 25 mL 3.5 mL 0.05 mL 0.25 mL 0.1 mL 2.5 mL

— 50% 1.4X SSC 0.5 mM 50 µg/mL 0.2% 0.5%

0.05 mL

50 µg/mL

Formamide SSC (20X, pH 4.5) EDTA (0.5 M in DEPC-H2O) tRNA (10 mg/mL) Tween 20 CHAPS (100 mg/mL in DEPC-H2O) (filter-sterilized before use) Heparin (50 mg/mL in DEPC-H2O)

Add all reagents in the order indicated. Stir until all ingredients are dissolved. Always prepare fresh for each experiment; store excess buffer for no longer than 24 h at 4°C.

Insulin solution for amphibians 50 mg insulin (from bovine pancreas; Sigma) 50 mL HCl (0.1 M) APBS To prepare 100 mL of insulin solution, weigh 50 mg of insulin in a beaker. Add 50 mL of 0.1 M HCl to dissolve the insulin. Add APBS to bring the volume to 100 mL. Sterilize by filtering through a 0.22-µm filter. Store as 1-mL aliquots at –20°C.

Intra-pipette solution 122 mM CsCl 20 mM tetraethylammonium chloride 3 mM Na2ATP 10 mM HEPES-CsOH (pH 7.3)

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K/Na-phosphate buffer (67 mM, pH 7.4) MATERIALS Reagents 0.1 M KH2PO4 0.1 M Na2HPO4

METHOD 1. Mix one volume of the KH2PO4 stock solution with four volumes of the Na2HPO4 stock solution. 2. Dilute two volumes of the 0.1 M K-Na phosphate buffer from Step 1 with one volume of water (i.e., to a final concentration of 67 mM).

LB agar Agar (20 g/L) NaCl (10 g/L; Sigma-Aldrich S9625) Tryptone (10 g/L; BD 211705) Yeast extract (5 g/L; BD 212750) Add H2O to a final volume of 1 L. Adjust the pH to 7.0 with 5 N NaOH. Autoclave. Pour into Petri dishes (~25 mL per 100-mm plate).

LB-ampicillin agar plates Ampicillin, filter-sterilized (10 mg/mL stock) LB agar

Autoclave 1 L of LB agar. Cool to 55°C. Add 10 mL of ampicillin stock. Pour into Petri dishes (~25 mL per 100-mm plate).

LB (Luria-Bertani) liquid medium Reagent

Amount to add

H2O Tryptone NaCl Yeast extract

950 mL 10 g 10 g 5g

Combine the reagents and shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (~0.2 mL). Adjust the final volume of the solution to 1 L with H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. For solid medium, see the recipe entitled “Media containing agar or agarose.”

LBAT LB (Luria-Bertani) liquid medium Ampicillin (100 µg/mL)

Tetracycline (12.5 µg/mL)

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LBAT-agar plates Ampicillin stock solution, filter-sterilized (100 mg/mL) LB agar

Tetracycline stock solution, filter-sterilized (12.5 mg/mL) Autoclave 1 L of LB agar. Cool to 55°C. Add 1 mL of ampicillin stock (to a final concentration of 100 µg/mL) and 1 mL of tetracycline stock (to a final concentration of 12.5 µg/mL). Pour into Petri dishes (~25 mL/100-mm plate).

M199S Fetal bovine serum, heat-inactivated (10%) Medium 199 (Sigma M4530)

MAB (10X) Reagent

Amount to add

Final concentration (10X)

Maleic acid NaCl NaOH H2O

116 g 87 g 40 g 800 mL

1M 1.5 M

To make 1 L of 10X MAB, combine the above reagents, and adjust the pH to 7.5. Beware that this is a weak buffer; pH 7.5 is easily missed. A 10X stock of MAB is preferred to a 5X stock because it does not get contaminated as easily.

MABT Reagent

Final concentration

Maleic acid (pH 7.5) NaCl Tween 20

100 mM 150 mM 0.1% (v/v)

Methylene blue/azure II staining solution Azure II stock solution (1%, prepared in water) Methylene blue stock solution Mix the azure II stock solution 1:1 with the methylene blue stock. Filter before use. The mixture is stable for several months. The Azure II stock solution is stable for up to 12 mo at room temperature.

Methylene blue stock solution Borax (1%) Methylene blue (1%) The solution is stable for up to 12 mo at room temperature.

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Mowiol embedding medium Reagent

Amount to add (for 50 mL)

Final conc.

Glycerol, puriss p.a. H2O Mowiol 4-88 Tris-Cl (0.2 M, pH 8.5)

12 g 12 mL 4.8 g 24 mL

24% (w/v) — 9.6% (w/v) 0.1 M

METHOD 1. 2. 3. 4. 5.

Mix glycerol and Mowiol; dissolve with frequent agitation for 1 h at room temperature. Add H2O. Stir for 1 h at room temperature. Add Tris-Cl. Incubate for 2 h at 50°C with occasional stirring (e.g., for 2 min every 20 min). Centrifuge at 5000g for 15 min. Store 3-mL aliquots of the supernatant for up to 12 mo at –20°C. Mowiol often does not dissolve completely.

NTMT for rabbit Reagent

Amount to add (for 400 mL)

Final concentration

Levamisole (1 M) MgCl2 (1 M) NaCl (5 M) Tris-Cl (1 M) Tween 20 DEPC-H2O

0.8 mL 20 mL 8 mL 40 mL 0.4 mL to 400 mL

2 mM 50 mM 100 mM 100 mM 0.1%

Add all reagents to ~300 mL DEPC-H2O. Mix. Adjust the volume to 400 mL with DEPC-H2O. Store NTMT for no more than several days at room temperature.

Osmium tetroxide stock solution (2%) MATERIALS Reagents Osmium tetroxide (osmium [VIII] tetroxide)

Equipment Aluminum foil Bottle, brown, wide-necked, sealable with a ground-glass stopper Box, polystyrene Fume hood Gloves, rubber Goggles Metal rod Ultrasonic bath

METHOD Perform all steps in a fume hood. Wear rubber gloves and goggles. 1. Pour 50 mL of H2O into a wide-necked brown glass bottle that can be sealed by a ground-glass stopper. 2. Place the bottle in a polystyrene box. 3. Submerge a glass ampoule containing 1 g of osmium oxide in the H2O. 4. Break the ampoule with a metal rod. 5. Stopper the bottle. Incubate for 10 min in an ultrasonic bath. 6. Wrap the bottle with aluminum foil. Incubate overnight in the fume hood to allow the osmium oxide to dissolve completely. Stored refrigerated at 4°C for up to 6 wk. www.cshprotocols.org

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Paraformaldehyde fixation solution (4%) Reagent

Amount to add

Final concentration

Paraformaldehyde stock solution (16%) DEPC-PBS (10X), diluted to 1X

50 mL 150 mL

4% 0.75X

Add the paraformaldehyde stock solution to 130 mL of 1X DEPC-PBS while stirring. If necessary, adjust the pH to 7.4. Add 1X DEPC-PBS to a final volume of 200 mL. Store aliquots (5-, 10-, or 20-mL) for up to 1 yr at –20°C.

Paraformaldehyde/glutaraldehyde fixation solution Reagent

Amount to add

Final concentration

Glutaraldehyde (25%)

60 mL 60 mL 450 mL to 1000 mL

1.5% 1.5% 0.09 M —

Paraformaldehyde stock solution (25%) Sodium phosphate buffer (0.2 M)

H2O

Add glutaraldehyde and paraformaldehyde to freshly prepared 0.2 M sodium phosphate buffer while stirring constantly. Add H2O to a final volume of 1000 mL. Store aliquots (5-, 10-, 20-, or 50-mL) for up to 1 yr at –20°C. Thaw aliquots immediately before use; discard the unused portion.

Paraformaldehyde stock solution (16%) Add 8 g of paraformaldehyde to 50 mL of water. Heat to 60°C-65°C, until the solution turns milky. Add 1 N NaOH dropwise while stirring until the solution clears. Cool to room temperature. Filter through a paper filter.

Paraformaldehyde stock solution (25%) Add 15 g of paraformaldehyde to 60 mL of water. Heat to 60°C-65°C, until the solution turns milky. Add 1 N NaOH dropwise while stirring until the solution clears. Cool to room temperature. Filter through a paper filter.

Paramecium culture medium MATERIALS Reagents

β-sitosterol (4 mg/mL, prepared in 100% ethanol) Store in the dark at 4°C. Feeding bacteria Wheat grass medium

Equipment Incubator preset to 27°C or 35°C (see Step 2) Inoculation loop

METHOD 1. Inoculate wheat grass medium (standard or rich, as appropriate for the intended experiment) with fresh bacteria. Paramecium culture medium is termed standard or rich, depending on whether it is made with standard or rich wheat grass medium.

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2. Incubate without shaking for 12-18 h (i.e., overnight) at 27°C. If Bio Herbe de Blé was used in the standard wheat grass medium, incubate at 35°C. 3. Immediately before use, add β-sitosterol to a final concentration of 0.8 µg/mL.

Pasteur pipettes with customized opening diameters MATERIALS Equipment Alcohol burner, glass Pasteur pipettes (glass, long) Long pipettes usually have a longer conical part than the short variety. Pencil (diamond-point) Plastic card (e.g., credit card) Protective eyegear and gloves

METHOD This method produces the desired opening size by defined fracturing. Protect your eyes and hands against flying glass splinters. 1. Hold the pipette horizontally. 2. Make a vertical notch at the conical part of the pipette with a diamond pen. Vary the position of the notch to get different diameters in the resulting opening. 3. Use a solid ridge (e.g., a stiff plastic card such as a credit card) to crack the pipette in two. With some practice, a vertical cut end is obtained at the intended level of the pipette conus. 4. Flame-polish the sharp edge of the pipette using an alcohol burner.

PBS (10X) without potassium salt Reagent

Amount to add (for 1L)

Final (10X) concentration

NaCl Na2HPO4•2H2O NaH2PO4•2H2O

75.97 g 12.46 g 4.8 g

1.3 M 70 mM 30 mM

Combine all components in
8 g 137 mM 0.2 g2.7 mM 1.44 g 0.24 g

80 g 2g 10 mM 1.8 mM

1.37 M 27 mM 14.4 g 2.4 g

100 mM 18 mM

If necessary, PBS may be supplemented with the following: CaCl2•2H2O 0.133 g 1 mM 1.33 g 10 mM 0.10 g 0.5 mM 1.0 g 5 mM MgCl2•6H2O PBS can be made as a 1X solution or as a 10X stock. To prepare 1 L of either 1X or 10X PBS, dissolve the reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required) with HCl, and then add H2O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.

Plastic culture rings MATERIALS Reagents Ethanol (70%) Gelatin/BSA embedding medium

Equipment Box, sterile Pipettes, transfer, plastic, 3.5-mL Laboratory tissues Razor blade Vibratome

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METHOD 1. 2. 3. 4. 5.

Use a razor blade to excise a region from a plastic transfer pipette at which the inner diameter is 3-5 mm. Embed the pipette piece in gelatin/BSA embedding medium. Cut the pipette piece into 300-µm-thick rings using the vibratome at very low speed. Sterilize the rings by washing in 70% ethanol. Wash the rings thoroughly in sterile water. Dry on tissues. Store rings in a sterile box until use.

R10 medium RPMI medium 1640 (GIBCO/Invitrogen) 10% fetal bovine serum (FBS) 100 U/mL penicillin (Multicell) 100 µg/mL streptavidin (Multicell) 2 mM L-glutamine (Multicell) 20 mM HEPES (Sigma) Adjust pH to 7.0.

R20 medium RPMI medium 1640 (GIBCO/Invitrogen) 20% fetal bovine serum (FBS) 100 U/mL of penicillin (Multicell) 100 µg/mL streptavidin (Multicell) 2 mM L-glutamine (Multicell) 20 mM HEPES (Sigma) Adjust pH to 7.0.

Rabbit embryo culture medium (RECM) Ham’s F10 medium with Phenol Red, NaHCO3, and stable glutamine (e.g., Biochrom FG0715) Penicillin (50 U/mL) Streptomycin (50 µg/mL) Fetal calf serum (FCS; 20%) (optional, as per the requirements of the individual experiment)

SF-AMEM 24 mL H2O (sterile; Merck) 1 mL antibiotic-antimycotic solution (AA solution; Sigma) 1 mL glutamine (Invitrogen) 1 mL insulin solution for amphibians 100 µL gentamicin (50 mg/mL; Sigma) 73 mL 1X MEM (Invitrogen) Prepare the medium in a sterile reagent bottle (e.g., a 75-cm2 tissue culture flask). Combine the reagents in the order listed. The resulting culture medium is serum-free MEM adjusted to amphibian osmolarity.

Small-bore pipettes MATERIALS Equipment Bunsen burner

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Forceps, small Pencil, diamond-point Tubes, capillary, borosilicate glass, 10-cm length, 1-mm OD, 0.5-mm ID (e.g., Clark GC 150-T10 or equivalent)

METHOD Small-bore pipettes are used for embryo transport and to inject embryos in the genital tract. 1. Hold a glass capillary tube at both ends. Heat at the center of the capillary by rotating the capillary in (or near) the flame of the Bunsen burner until the capillary melts. 2. Immediately after the capillary starts to melt, quickly remove the capillary from the flame. Pull it straight to obtain a pulled capillary in the center region. 3. Cut the capillary in the pulled region with the diamond-point pencil. These capillaries can be used for most embryo transport applications and transfer of embryos to the uterotubal junction. 4. If required, prepare capillaries for transferring embryos to the oviduct: i. Heat the capillary (from Step 3) with the tip of the flame. ii. Approximately 1 cm from the tip, bend the capillary to an angle of 20° relative to the long axis.

Sodium phosphate buffer (0.2 M) MATERIALS Reagents Na2HPO4•2H2O NaH2PO4•H2O

METHOD 1. Dissolve 35.61 g of Na2HPO4•2H2O and 27.6 g of NaH2PO4•H2O separately in H2O. 2. Adjust the volume of each solution to 1000 mL. Store the stock solutions for up to 6 mo at 4°C. 3. To prepare the buffer, mix the stock solutions as follows: i. For 50 mL: 38.5 mL of Na2HPO4•2H2O and 11.5 mL of NaH2PO4•H2O. ii. For 100 mL: 77 mL of Na2HPO4•2H2O and 23 mL of NaH2PO4•H2O. iii. For 500 mL: 385 mL of Na2HPO4•2H2O and 115 mL of NaH2PO4•H2O. iv. For 1000 mL: 770 mL of Na2HPO4•2H2O and 230 mL of NaH2PO4•H2O. 4. Check the pH of the solution at room temperature. If necessary, adjust the pH to 7.3.

SSC For a 20X solution: Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 mL of H2O. Adjust the pH to 7.0 with a few drops of a 14 N solution of HCl. Adjust the volume to 1 L with H2O. Dispense into aliquots. Sterilize by autoclaving. The final concentrations of the ingredients are 3.0 M NaCl and 0.3 M sodium citrate.

Subbed slides MATERIALS Reagents Chromium potassium sulfate Gelatin (Sigma) Haemo-sol (warm)

Equipment Hot plate Slide racks Slides www.cshprotocols.org

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METHOD 1. Dissolve 5 g of gelatin and 0.5 g of chromium potassium sulfate in 500 mL of hot (80ºC) H2O. Stir on a hot plate until dissolved (~2 h). 2. Place slides in slide racks and wash in a warm solution of Haemo-sol. 3. Rinse slides well in H2O. 4. Dip slides in the gelatin solution prepared in Step 1. 5. Cover slides loosely and dry overnight.

Suction/injection pipettes MATERIALS Reagents Fluoric acid (2%)

Equipment Abrasive film, 0.3-µm (e.g., Thomas Scientific 6775E54) Capillary tubes, borosilicate glass, 10-cm length, 1-mm OD, 0.5-mm ID (e.g., Clark or equivalent) Container (clean, airtight) Microforge, equipped with micrometer scale eyepiece (e.g., deFonbrune; Alcatel or equivalent) Micropipette puller (e.g., Campden Instruments Ltd. or equivalent) Pipette grindstone Rubber tubing Syringe, 10-mL

METHOD 1. Switch on the micropipette puller. Program with the following recommended settings: Prepull heating time: 048 Initial pull force: 958 Main pull force: 395 Main pull delay: 062 Heater control: 140 2. Place the capillary tube in the micropipette puller according to the manufacturer instructions. Press “Load” and “Pull” to obtain a pulled capillary. 3. Clamp the pulled capillary into the microforge. 4. Turn on the microforge. Guide the pulled capillary horizontally relative to the heating filament (i.e., at the level of the small glass ball) and slightly below it. 5. Using the 20X objective and the micrometer scale eyepiece, focus on a region with a 7-µm OD. 6. Heat the filament to ~60°C. 7. Move the pulled capillary toward the heating filament. When the capillary reaches the small glass ball, stop heating. Raise the pulled capillary quickly. This should cut the pulled capillary; verify that the capillary is cut straight. 8. Bevel the capillary tip with a grindstone and abrasive film to produce a tip with an OD of 7-10 µm. 9. Using a syringe with attached rubber tubing, rinse the beveled injection capillary for a few seconds with 2% fluoric acid to remove the glass dust. Rinse well with water. 10. Guide the pulled capillary vertically relative to the heating filament. Move the heating filament close to the capillary (e.g., 2-5 mm from the tip of the pipette). 11. Carefully heat the filament to ~70°C until the capillary begins to bend. Stop heating when the desired angle is obtained. The angle will vary according to the type of micromanipulator used. 12. Store suction/injection pipettes in a clean airtight container until use.

TBSTEM 10 mM EGTA 2 mM MgCl2

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0.15 M NaCl 10 mM Tris Adjust the pH to 7.4. Add Tween 20 (1%, v/v). If the buffer is to be used for immunostaining of Ca2+-binding structures, omit the EGTA and Mg2+ (i.e., just use Tris-buffered saline containing 1% Tween 20).

Tris-Cl Tris base HCl To prepare a 1 M solution, dissolve 121.1 g of Tris base in 800 mL of H2O. Adjust the pH to the desired value by adding concentrated HCl. pH

HCl

7.4 7.6 8.0

70 mL 60 mL 42 mL

Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 L with H2O. Dispense into aliquots and sterilize by autoclaving. If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature-dependent and decreases ~0.03 pH units for each 1°C increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5°C, 25°C, and 37°C, respectively.

Tris diluent 88 mM citric acid (Sigma C2404) 47 mM D-(+)-glucose (Sigma G6152) 0.25 M Tris base (Trizma base; Sigma T6066)

Trypsin-calcium 0.1% CaCl2 0.05% trypsin Adjust the pH to 7.8.

Tungsten needles MATERIALS Reagents Potassium nitrite (KNO2) solution, saturated, prepared in water Alternatively, use 1 N NaOH.

Equipment Clips, alligator Dish, porcelain Ethanol burner Forceps, fine Pasteur pipettes with customized opening diameters Prepare the pipettes such that the opening of the resulting capillary pipette will hold the sharpened tungsten needles. Power cable

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Stereomicroscope (e.g., Stemi SV11; Zeiss) Transformer, alternating current, capable of generating 4-8 V and up to 5 A Tripod Wire, platinum Wire, tungsten, 0.1- to 0.2-mm diameter

METHOD 1. Clamp a 3- to 4-cm piece of tungsten wire in an alligator clip fixed to a tripod. Clamp a 3- to 4-cm piece of platinum wire in another alligator clip fixed to a tripod. 2. Submerge the tip of the platinum wire in a porcelain dish filled with the saturated KNO2 solution (or 1 N NaOH). 3. Connect the power cables of the alligator clips to the transformer. 4. While monitoring under a stereomicroscope, dip the tip of the tungsten wire repeatedly in the KNO2 solution to obtain a 2- to 3-mm-long conus with the desired sharpness. 5. Using fine forceps, place the sharpened tungsten needle into the opening of the capillary pipette. 6. Using an ethanol burner, melt the glass around the needle, fixing it into place. 7. Wash the tungsten needle extensively in water to remove any residual salt crystals. Blunt or damaged needles can be resharpened by flame-polishing using an ethanol burner. Although needle tips are less smooth after flame-polishing (compared with the procedure above), flame-polishing is acceptable as a fast and convenient method during embryo dissection.

Tyramine HCl stock Reagent

Amount to add

DMF (dimethylformamide, anhydrous) Triethylamine Tyramine HCl

1 mL 10 µL 10 mg

Vector Blue solution 0.1M Tris-Cl (pH 8.2) 0.75% Vector Blue Reagent 1 0.8% Vector Blue Reagent 2 0.5% Vector Blue Reagent 3 Vector Blue Reagents 1, 2, and 3 are from the Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories). Vector Blue solution should be prepared within hours of use.

Wash medium for ELISPOT assay RPMI medium 1640 (GIBCO/Invitrogen) 2% fetal bovine serum (FBS) 100 U/mL penicillin (Multicell) 100 µg/mL streptavidin (Multicell)

Washing solution for rabbit DEPC-PBS (10X), diluted to 1X Normal goat serum (NGS) (0.1%, v/v) Triton X-100 (0.1%, v/v) Complement-deactivate NGS by incubation for 1 h at 60°C. Store 2-mL aliquots of NGS for up to 12 mo at –20°C.

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Wheat grass medium Quantity (for 20 L) Reagent

Rich medium

Standard medium

Na2HPO4•12H2O (50 mM)

1L 1L to 20 L

1L 200 mL to 20 L

Wheat grass stock solution

Water, Millipore-filtered

Autoclave for 20 min at 120°C. For rich medium, only use wheat grass stock solution prepared with wheat grass powder.

Wheat grass stock solution MATERIALS Reagents Wheat grass powder (Pines International, Inc.) Bio Herbe de Blé (l’Arbre de Vie) can also be used, but is not suitable for rich Paramecium culture medium. Water, Millipore-filtered

Equipment Autoclave Filter, cotton gauze Flask, glass, 1-L Flasks, glass, 200-mL Funnel Hot plate

METHOD 1. 2. 3. 4. 5.

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Add 50 g of wheat grass powder to 750 mL of Millipore-filtered water in a glass flask. Boil for 15 min. Filter the suspension through a thick layer of cotton gauze. Press to recover as much fluid as possible. Adjust the volume to 1 L with Millipore-filtered water. Distribute the medium into five 200-mL flasks. Autoclave for 20 min at 120°C. Store the sterilized stock solution for weeks at 4°C-5°C.

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Appendix 2: Cautions [NOTE: For reagents marked with the symbol not listed below, please consult the manufacturer’s Material Safety Data Sheet for further information. Researchers using the procedures in these protocols contained in this issue of CSH Protocols do so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in these protocols and has no liability in connection with the use of these materials. Materials used in these protocols may be considered hazardous and should be used with caution.]

to the eyes. Wear appropriate gloves and safety goggles. Do not breathe the dust.

Acetic acid (concentrated) must be handled with great care. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and goggles. Use in a chemical fume hood.

CoCl2 (Cobalt chloride) is toxic, is a possible carcinogen, and is dangerous to the environment. It can cause burns and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.

AEC (Aminoethylcarbazole) may be harmful by inhalation, ingestion, and skin absorption. Wear appropriate gloves and safety glasses.

CsCl (Cesium chloride) may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.

Ampicillin may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses and use in a chemical fume hood.

CsOH (Cesium hydroxide) may be harmful by inhalation, ingestion, or skin absorption. It is extremely destructive to the mucous membranes and upper respiratory tract; inhalation may be fatal. Do not breathe the dust. Wear appropriate gloves and safety glasses and always use in a chemical fume hood.

Araldite may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Do not breathe the vapor. BM purple may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and glasses.

DAB (3,3′-Diaminobenzidine tetrahydrochloride) is a carcinogen. Handle with extreme care. Avoid breathing vapors. Wear appropriate gloves and safety glasses and use in a chemical fume hood.

BrdU (Bromodeoxyuridine, 5-Bromo-2′-deoxyuridine, 5-Bromodeoxyuridine) is a mutagen. It may be harmful by inhalation, ingestion, or skin absorption. It may cause irritation. Avoid breathing the dust. Wear appropriate gloves and safety glasses and always use in a chemical fume hood.

DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride) is a possible carcinogen. It may be harmful by inhalation, ingestion, or skin absorption. It may also cause irritation. Avoid breathing the dust and vapors. Wear appropriate gloves and safety glasses and use in a chemical fume hood.

Buserelin may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Do not breathe the dust.

DEPC (Diethyl pyrocarbonate) is a potent protein denaturant and is a suspected carcinogen. Aim bottle away from you when opening it; internal pressure can lead to splattering. Wear appropriate gloves and lab coat. Use in a chemical fume hood.

CaCl2 (Calcium chloride) is hygroscopic and may cause cardiac disturbances. It may be harmful by inhalation, ingestion, or skin absorption. Do not breathe the dust. Wear appropriate gloves and safety goggles.

Diazepam is toxic, a mutagen, and a teratogen. It is harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Do not breathe the dust.

CHAPS (3-[(3-Cholamidopropyl)dimethyl-ammonio]-1propanesulfonate) is an irritant and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Citifluor mountant media is highly alkaline and irritating to the eyes and the skin. It may be harmful by inhalation, ingestion, or skin absorption. It poses a risk of serious damage to the eyes. Wear appropriate gloves and safety goggles. Do not breathe the vapor.

DMF (N,N-Dimethylformamide, dimethylformamide, HCON[CH3]2) is a possible carcinogen and is irritating to the eyes, skin, and mucous membranes. It can exert its toxic effects through inhalation, ingestion, or skin absorption. Chronic inhalation can cause liver and kidney damage. Wear appropriate gloves and safety glasses. Use in a chemical fume hood.

Citric acid is an irritant and may be harmful by inhalation, ingestion, or skin absorption. It poses a risk of serious damage

DMSO (Dimethyl sulfoxide) may be harmful by inhalation or skin absorption. Wear appropriate gloves and safety glasses.

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H2O2 (Hydrogen peroxide) is corrosive, toxic, and extremely damaging to the skin. It may be harmful by inhalation, ingestion, and skin absorption. Wear appropriate gloves and safety glasses. Use only in a chemical fume hood.

Use in a chemical fume hood. DMSO is also combustible. Store in a tightly closed container. Keep away from heat, sparks, and open flame. DPX is composed of Distyrene, a plasticizer, and xylene and is commercially available. Follow the manufacturer’s guidelines for handling DPX.

HCl (Hydrochloric acid, Hydrochloride) is volatile and may be fatal if inhaled, ingested, or absorbed through the skin. It is extremely destructive to mucous membranes, upper respiratory tract, eyes, and skin. Wear appropriate gloves and safety glasses. Use with great care in a chemical fume hood. Wear goggles when handling large quantities.

Dry ice (Carbon dioxide; CO2) CO2 (carbon dioxide; dry ice) in all forms may be fatal by inhalation, ingestion, or skin absorption. In high concentrations, it can paralyze the respiratory center and cause suffocation. Use only in well-ventilated areas. In the form of dry ice, contact with carbon dioxide can also cause frostbite. Do not place large quantities of dry ice in enclosed areas such as cold rooms. Wear appropriate gloves and safety goggles.

Heparin is an irritant and may act as an anticoagulant subcutaneously or intravenously. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.

Dyes Follow manufacturer’s safety guidelines.

Histoclear is flammable and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Keep away from heat, sparks, and open flame.

EDTA (Ethylenediamenetetraacetic acid) may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Severe overexposure can result in death.

Hoechst 33342 (sometimes called bisbenzimide) may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses and use in a chemical fume hood. Do not breathe the dust.

Fast Green is a carcinogen and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses and always use in a chemical fume hood.

IPBA (p-Iodophenylboronic acid) may be harmful by inhalation, ingestion, or skin absorption, and is flammable. Wear appropriate gloves and safety goggles and use only in a chemical fume hood.

Fluorescein isothiocyanate (FITC) may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.

IPTG (Isopropyl-β-D-thiogalactopyranoside) may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.

Fluoric acid is extremely hazardous in both liquid and vapor forms. It is corrosive and poisonous and may be fatal. It is harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles and use in a chemical fume hood. Do not breathe the vapor. Keep away from heat, sparks, and open flame.

Isofluorane (Isoflurane) is an irritant and may be harmful by inhalation, ingestion, or skin absorption. Chronic exposure may be harmful. Wear appropriate gloves and safety glasses.

Formaldehyde (HCOH) is highly toxic and volatile. It is also a carcinogen. It is readily absorbed through the skin and is irritating or destructive to the skin, eyes, mucous membranes, and upper respiratory tract. Avoid breathing the vapors. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood. Keep away from heat, sparks, and open flame.

KCl (Potassium chloride) may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. KOH and KOH/methanol (Potassium hydroxide) can be highly toxic. It may be harmful by inhalation, ingestion, or skin absorption. Solutions are caustic and should be handled with great care. Wear appropriate gloves.

Formamide is teratogenic. The vapor is irritating to the eyes, skin, mucous membranes, and upper respiratory tract. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood when working with concentrated solutions of formamide. Keep working solutions covered as much as possible.

Levamisole (Levamisol HCl, Levamisol hydrochloride) is toxic if ingested. It may be harmful by inhalation or skin absorption. Wear appropriate gloves and safety glasses. Liquid nitrogen (LN2) can cause severe damage due to its extreme temperature. Handle frozen samples with extreme caution. Do not breathe the vapors. Seepage of liquid nitrogen into frozen vials that are immersed in liquid nitrogen can cause the vials to explode when they are removed. Use vials with O-rings when possible. Wear cryo-mitts and a face mask. Do not allow the liquid nitrogen to spill onto clothing. Do not breathe the vapors.

Glutaraldehyde is toxic. It is readily absorbed through the skin and is irritating or destructive to the skin, eyes, mucous membranes, and upper respiratory tract. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood.

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depression and sedation and presents a risk to the unborn child. Do not breathe the dust. Wear appropriate gloves and safety glasses and use in a chemical fume hood.

Lysozyme is caustic to mucous membranes. Wear appropriate gloves and safety glasses. Maleic acid is toxic and harmful by inhalation, ingestion, or skin absorption. Reaction with water or moist air can release toxic, corrosive, or flammable gases. Do not breathe the vapors or dust. Wear appropriate gloves and safety glasses.

Phenol is extremely toxic, highly corrosive, and can cause severe burns. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves, goggles, and protective clothing. Always use in a chemical fume hood. Rinse any areas of skin that come in contact with phenol with a large volume of water and wash with soap and water; do not use ethanol!

Meloxicam is toxic and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles. Do not breathe the dust.

Potassium nitrite (KNO2) is toxic and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses and use in a chemical fume hood. Do not breathe the dust.

Methanol (MeOH, H3COH) is poisonous and can cause blindness. It may be harmful by inhalation, ingestion, or skin absorption. Adequate ventilation is necessary to limit exposure to vapors. Avoid inhaling these vapors. Wear appropriate gloves and goggles. Use only in a chemical fume hood.

Propylene oxide is highly flammable, toxic, and may be carcinogenic. High concentrations are extremely destructive to the mucous membranes and upper respiratory tract. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses and use only in a chemical fume hood. Keep away from heat, sparks, and open flame.

Methylene blue is irritating to the eyes and skin. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. NaOH (Sodium hydroxide) and solutions containing NaOH are highly toxic and caustic and should be handled with great care. Wear appropriate gloves and a face mask. All concentrated bases should be handled in a similar manner.

Proteinase K is an irritant and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.

NiCl2 (Nickel chloride) is toxic and may be harmful by inhalation, ingestion, or skin absorption. Do not breathe the dust. Wear appropriate gloves and safety glasses.

Resins are suspected carcinogens. The unpolymerized components and dusts may cause toxic reactions, including contact allergies with long-term exposure. Avoid breathing the vapors and dusts. Wear appropriate gloves and safety goggles and always use in a chemical fume hood. Sensitivity to these chemicals may develop with repeated contact.

OCT is composed of polyvinyl alcohol, polyethylene glycol, and dimethyl benzyl ammonium chloride. Follow the manufacturer’s guidelines for handling OCT. Osmium tetroxide (Osmic acid, OsO4) is highly toxic if inhaled, ingested, or absorbed through the skin. Vapors can react with corneal tissues and cause blindness. There is a possible risk of irreversible effects. Wear appropriate gloves and safety goggles and always use in a chemical fume hood. Do not breathe the vapors.

Saponin is an irritant and may be harmful by inhalation, ingestion, or skin absorption. Do not breathe the dust. Wear appropriate gloves and safety glasses and use in a chemical fume hood. SDS (Sodium dodecyl sulfate) is toxic, an irritant, and poses a risk of severe damage to the eyes. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles. Do not breathe the dust.

Oxygen (O2) presents a fire and explosion hazard. The gas is heavier than air and is a strong oxidant. Vapors may cause dizziness or asphyxiation without warning. Keep away from heat, sparks, and open flame.

Sodium citrate Related to citric acid. Citric acid is an irritant and may be harmful by inhalation, ingestion, or skin absorption. It poses a risk of serious damage to the eyes. Wear appropriate gloves and safety goggles. Do not breathe the dust.

Oxytetracycline may be harmful by inhalation, ingestion, or skin absorption. Do not breathe the dust. Paraformaldehyde is highly toxic and may be fatal. It may be a carcinogen. It is readily absorbed through the skin and is extremely destructive to the skin, eyes, mucous membranes, and upper respiratory tract. Avoid breathing the dust or vapor. Wear appropriate gloves and safety glasses and use in a chemical fume hood. Keep away from heat, sparks, and open flame.

Streptomycin is toxic and a suspected carcinogen and mutagen. It may cause allergic reactions. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Sulfamerazine is an irritant and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.

Pentobarbital sodium is toxic and may be harmful by inhalation, ingestion, or skin absorption. It can induce respiratory

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Tetracycline may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses and use in a chemical fume hood.

safety glasses and use in a chemical fume hood. Keep away from heat, sparks, and open flame. Tris base may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.

Tetraethylammonium chloride (TEAC) may cause allergic skin reaction and may be harmful by inhalation, ingestion, or skin absorption. It is irritating to the mucous membranes and upper respiratory tract. Do not breathe the dust. Wear appropriate gloves and safety glasses.

Triton X-100 causes severe eye irritation and burns. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles.

Tricaine (Tricaine methanesulfonate) is an irritant and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses.

Trypsin may cause an allergic respiratory reaction. It may be harmful by inhalation, ingestion, or skin absorption. Do not breathe the dust. Wear appropriate gloves and safety goggles. Use with adequate ventilation.

Triethylamine (TEN) is highly toxic and flammable. It is extremely corrosive to the mucous membranes, upper respiratory tract, eyes, and skin. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and

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Virkon disinfectant is an irritant and may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles. Do not breathe the dust.

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