Work in the Lee lab was supported by the National Institute of General Medical Sciences under award number 1R15GM144861 and the National Institute for Child and Human Development under award number 1R15HD104115, both of the National Institutes of Health. DA was supported by the UML Kennedy College of Sciences Science Scholars program. NW was supported by a UML Honors College Fellowship and a UMLSAMP fellowship (funded by the National Science Foundation under grant number HRD-1712771). We thank A. Gartner for RAD-51 antibody. All C. elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by the Office of Research Infrastructure Programs of the National Institutes of Health under award numberP40 OD010440.

<ol>
	<li>Newman, D. L., Wright, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis:+A+play+in+three+acts,+starring+DNA+sequence.">Meiosis: A play in three acts, starring DNA sequence.</a> CourseSource. 4, (2017).</li><li>Ohkura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis:+An+overview+of+key+differences+from+mitosis.">Meiosis: An overview of key differences from mitosis.</a> Cold Spring Harbor Perspectives in Biology. 7 (5), 015859 (2015).</li><li>Hassold, T., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nature Reviews Genetics. 2 (4), 280-291 (2001).</li><li>Keeney, S., Giroux, C. N., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis-specific+DNA+double-strand+breaks+are+catalyzed+by+Spo11,+a+member+of+a+widely+conserved+protein+family.">Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family.</a> Cell. 88 (3), 375-384 (1997).</li><li>Rabitsch, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetochore+recruitment+of+two+nucleolar+proteins+is+required+for+homolog+segregation+in+meiosis+I.">Kinetochore recruitment of two nucleolar proteins is required for homolog segregation in meiosis I.</a> Developmental Cell. 4 (4), 535-548 (2003).</li><li>Brown, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+misconceptions+in+meiosis+shown+by+students+responding+to+an+advanced+level+practical+examination+question+in+biology.">Some misconceptions in meiosis shown by students responding to an advanced level practical examination question in biology.</a> Journal of Biological Education. 24 (3), 182-186 (1990).</li><li>Kalas, P., O'Neill, A., Pollock, C., Birol, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+meiosis+concept+inventory.">Development of a meiosis concept inventory.</a> CBE-Life Sciences Education. 12 (4), 655-664 (2013).</li><li>McDonnel, L. M., Klenz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teaching+genetic+linkage+and+recombination+through+mapping+with+molecular+markers.">Teaching genetic linkage and recombination through mapping with molecular markers.</a> CourseSource. 2, (2015).</li><li>Wright, L. K., Cortez, P., Franzen, M. A., Newman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teaching+meiosis+with+the+DNA+triangle+framework:+A+classroom+activity+that+changes+how+students+think+about+chromosomes.">Teaching meiosis with the DNA triangle framework: A classroom activity that changes how students think about chromosomes.</a> Biochemistry and Molecular Biology Education. 50 (1), 44-54 (2021).</li><li>Shortlidge, E. E., Bangera, G., Brownell, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Each+to+their+own+CURE:+Faculty+who+teach+course-based+undergraduate+research+experiences+report+why+you+too+should+teach+a+CURE.">Each to their own CURE: Faculty who teach course-based undergraduate research experiences report why you too should teach a CURE.</a> Journal of Microbiology &amp; Biology Education. 18 (2), 18 (2017).</li><li>Auchincloss, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+course-based+undergraduate+research+experiences:+A+meeting+report.">Assessment of course-based undergraduate research experiences: A meeting report.</a> CBE-Life Sciences Education. 13 (1), 29-40 (2014).</li><li>Lee, T. W., Carpenter, B. S., Birol, O., Katz, D. J., Schmeichel, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pipeline+CURE:+An+iterative+approach+to+introduce+all+students+to+research+throughout+a+biology+curriculum.">The pipeline CURE: An iterative approach to introduce all students to research throughout a biology curriculum.</a> CourseSource. 6, (2019).</li><li>Lu, F. -. M., Eliceiri, K. W., Stewart, J., White, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WormClassroom.org:+an+inquiry-rich+educational+web+portal+for+research+resources+of+Caenorhabditis+elegans.">WormClassroom.org: an inquiry-rich educational web portal for research resources of Caenorhabditis elegans.</a> CBE Life Sciences Education. 6 (2), 98-108 (2007).</li><li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+window+into+biology:+A+primer+on+Caenorhabditis+elegans.">A Transparent window into biology: A primer on Caenorhabditis elegans.</a> Genetics. 200 (2), 387-407 (2015).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+I:+yeast,+Drosophila+and+C.+elegans.+An+Introduction+to+Caenorhabditis+elegans.">Biology I: yeast, Drosophila and C. elegans. An Introduction to Caenorhabditis elegans.</a> JoVE Science Education Database. , (2022).</li><li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science+Education+Database.+Biology+I:+yeast,+Drosophila+and+C.+elegans.+C.+elegans+Maintenance.">Science Education Database. Biology I: yeast, Drosophila and C. elegans. C. elegans Maintenance.</a> JoVE Science Education Database. , (2022).</li><li>Lee, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web+resources+for+C.+elegans+studies.">Web resources for C. elegans studies.</a> WormBook. , (2005).</li><li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science+Education+Database.+Biology+I:+yeast,+Drosophila+and+C.+elegans.+RNAi+in+C.+elegans.">Science Education Database. Biology I: yeast, Drosophila and C. elegans. RNAi in C. elegans.</a> JoVE Science Education Database. , (2022).</li><li>Meneely, P. M., Dahlberg, C. L., Rose, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+worms:+Caenorhabditis+elegans+as+a+model+organism.">Working with worms: Caenorhabditis elegans as a model organism.</a> Current Protocols Essential Laboratory Techniques. 19 (1), 35 (2019).</li><li>Lemons, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inquiry-based+approach+to+study+the+synapse:+Student-driven+experiments+using+C.+elegans.">An inquiry-based approach to study the synapse: Student-driven experiments using C. elegans.</a> Journal of Undergraduate Neuroscience Education. 15 (1), 44-55 (2016).</li><li>Kowalski, J. R., Hoops, G. C., Johnson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+a+collaborative+series+of+classroom-based+undergraduate+research+experiences+spanning+chemical+biology,+biochemistry,+and+neurobiology.">Implementation of a collaborative series of classroom-based undergraduate research experiences spanning chemical biology, biochemistry, and neurobiology.</a> CBE-Life Sciences Education. 15 (4), 55 (2016).</li><li>Mordacq, J., Drane, D., Swarat, S., Lo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+and+teaching:+Development+of+course-based+undergraduate+research+experiences+using+a+design-based+approach.">Research and teaching: Development of course-based undergraduate research experiences using a design-based approach.</a> Journal of College Science Teaching. 46 (4), (2017).</li><li>Palmisano, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+laboratory+module+that+explores+RNA+interference+and+codon+optimization+through+fluorescence+microscopy+using+Caenorhabditis+elegans.">A laboratory module that explores RNA interference and codon optimization through fluorescence microscopy using Caenorhabditis elegans.</a> bioRxiv. , 344069 (2021).</li><li>Hillers, K. J., Villeneuve, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+meiotic+recombination+in+Caenorhabditis+elegans.">Analysis of meiotic recombination in Caenorhabditis elegans.</a> Meiosis. 557, 77-97 (2009).</li><li>Phillips, C. M., McDonald, K. L., Dernburg, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+analysis+of+meiosis+in+Caenorhabditis+elegans.">Cytological analysis of meiosis in Caenorhabditis elegans.</a> Meiosis. 558, 171-195 (2009).</li><li>Gervaise, A. L., Arur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+analysis+of+active+ERK+in+the+C.+elegans+germline.">Spatial and temporal analysis of active ERK in the C. elegans germline.</a> Journal of Visualized Experiments. (117), e54901 (2016).</li><li>Shakes, D. C., Miller, D. M., Nonet, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+microscopy.">Immunofluorescence microscopy.</a> Methods in Cell Biology. 107, 35-66 (2012).</li><li>Hillers, K. J., Jantsch, V., Martinez-Perez, E., Yanowitz, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis.">Meiosis.</a> Wormbook. , (2017).</li><li>Hunter, A. -. B., Seymour, E., Laursen, S., Thiry, H., Melton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Undergraduate Research in the Sciences: Engaging Students in Real Science. , (2010).</li><li>Wei, C. A., Woodin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Undergraduate+research+experiences+in+biology:+Alternatives+to+the+apprenticeship+model.">Undergraduate research experiences in biology: Alternatives to the apprenticeship model.</a> CBE-Life Sciences Education. 10 (2), 123-131 (2011).</li><li>Elgin, S. C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+from+a+convocation:+Integrating+discovery-based+research+into+the+undergraduate+curriculum.">Insights from a convocation: Integrating discovery-based research into the undergraduate curriculum.</a> CBE-Life Sciences Education. 15 (2), (2016).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+Elegant.">Maintenance of C. Elegant.</a> WormBook. , (2006).</li><li>Jaramillo-Lambert, A., Ellefson, M., Villeneuve, A. M., Engebrecht, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+timing+of+S+phases,+X+chromosome+replication,+and+meiotic+prophase+in+the+C.+elegans+germ+line.">Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line.</a> Developmental Biology. 308 (1), 206-221 (2007).</li><li>Stamper, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DSB-1,+a+protein+required+for+initiation+of+meiotic+recombination+in+Caenorhabditis+elegans,+illuminates+a+crossover+assurance+checkpoint.">Identification of DSB-1, a protein required for initiation of meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance checkpoint.</a> PLoS Genetics. 9 (8), 1003679 (2013).</li><li>Libuda, D. E., Uzawa, S., Meyer, B. J., Villeneuve, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+chromosome+structures+constrain+and+respond+to+designation+of+crossover+sites.">Meiotic chromosome structures constrain and respond to designation of crossover sites.</a> Nature. 502 (7473), 703-706 (2013).</li></ol>

The authors have no conflicts of interest to disclose. The content in this manuscript is solely the responsibility of the authors. It does not necessarily represent the official views of the National Institutes of Health or the National Science Foundation.

Sexual reproduction requires the creation of haploid gametes, which are produced via the specialized cell division of meiosis. C. elegans has become a popular model for the cytological study of meiosis due to its optical transparency, convenient germline anatomy, and powerful genetics28. Simple organismal experiments assessing fertility and embryonic lethality can be combined with molecular genetics to address many questions in the lab or the classroom. For example, because crossovers are essential for proper chromosome segregation, processes that disrupt their formation or resolution will generate aneuploid gametes. In turn, aneuploidy leads to inviable progeny, which can easily be assessed by counting progeny, or through the slightly more complicated method of determining embryonic lethality. Cytologically, a lack of crossovers will affect the numbers of DAPI-staining bodies observed during diakinesis. The robustness of DAPI staining and the ease of scoring makes this an ideal experiment to teach cytological techniques. The temporal-spatial layout of nuclei in the hermaphrodite germline provides a snapshot of each stage of meiosis at a single moment in time. Nuclei take about 54 hours to proceed from the distal mitotic end of the germline to the proximal end (for examples, see Jaramillo-Lambert et al.33, Stamper et al.34 and Libuda et al.35). This well-established timing of meiotic progress allows for pulse-chase labeling or DNA damaging experiments.
Because DAPI staining nearly always works, it serves as a useful technical control for fluorescence microscopy (and can provide satisfaction for microscopists-in-training). Antibody staining can be more variable and is a good measure of reproducibility between replicates. C.elegans gonads can be difficult to fix consistently, as this step requires a balance between preserving chromosomal structure while also allowing enough antibody diffusion. Fixing with formaldehyde best preserves chromosomal morphology, and preparing formaldehyde fresh from paraformaldehyde ampules has provided the most reproducible results. It is also possible to use formaldehyde prepared from formalin (37% aqueous formaldehyde and methanol). Fixation strength and timing may need to be empirically determined for each antibody. Alternative methods involve skipping the formaldehyde fixation in step 2.4 and, following freeze-crack, immersion in 100% ethanol at -20 &#176;C, or immersion in 100% methanol for 10 min followed by immersion in 100% acetone for 10 min at -20 &#176;C. These fix conditions allow better antibody penetration but will negatively affect tissue morphology (chromosomes will look blown-out and puffier).
Timing is very important for the dissection and fix steps outlined in step 2. Because the volume of dissection solution is so small, evaporation can impact the final fix concentration, which will cause variable antibody staining. An experienced C. elegans handler can prepare a slide in less than 2 min from the start (step 2.3) to fix (step 2.4). The main technical hurdle is the ability to pick animals into the drop of dissection solution and quickly dissect them. It can be easier to learn how to dissect in larger volumes. New trainees first start by picking animals into 50-200 &#181;L of dissection solution in a glass embryo staining dish (Figure 2D); the wider surface provides more room to maneuver when identifying an ideal needle position for good gonad extrusions, while the larger volume of liquid makes evaporation less of an issue. Once comfortable with the motions of dissecting, trainees start dissecting using only 50 &#181;L in the dish, then switch to dissecting in 20 &#181;L on a slide. Once dissecting on slides, trainees can quickly start reducing the amount of solution until they are quick enough at working in 4 &#181;L to make evaporation negligible. 
If the timing of dissection remains an issue, trainees can dissect in 8 &#181;L of the dissection solution during step 2.3. Then, during step 2.4, they can add 8 &#181;L of the fix solution, gently pipetting to mix thoroughly (watching closely to ensure that carcasses are not disrupted or pipetted away), and removing 8 &#181;L from the mixed solution. This alternative approach will result in the same final volume of 8 &#181;L and the same final fix concentration; this volume is important to ensure that carcasses contact both the coverslip and slide surface during the freeze-crack in step 2.5. If the timing of preparing each slide remains an issue even in larger dissection volumes, a suspension method for germline immunofluorescence protocol is described by Gervaise and Arur26.
The major limitation of using immunofluorescence to visualize proteins in situ is the availability of primary antibodies targeting a particular protein of interest. However, if a tagged version of the protein has been engineered, this protocol can be adapted to visualize the protein tag. Antibodies targeting common tags, like FLAG, HA, or GFP, are commonly available. Fluorescent tags like GFP can often be quenched by fixation steps, so it is recommended to use a primary antibody targeting GFP, rather than relying on the native fluorescence signal itself. Another limitation of this protocol is that it captures the germline within a single timeframe (although all stages of oogenesis will be represented within the germline). Therefore, this technique can miss dynamic changes that may occur as an oocyte progresses through oogenesis. However, the timing of gametogenesis has been well-studied in C. elegans; in a wild-type young adult, an oocyte takes about 60 h to progress from the distal tip of the germline (the mitotic zone) to diakinesis33. Therefore, a pulse chase or specific intervention like irradiation followed by a time course would allow for the observation of effects at different stages of meiosis.
In conclusion, this protocol describes C. elegans gonad dissection followed by DAPI and antibody staining for fluorescence microscopy. Dissection and fixing (step 2) takes 60-90 min, depending on the number of slides generated. Antibody staining (step 3) is mostly hands-off and can range from 7 h at minimum to 1.5 days, depending on antibody incubation times. Mounting (steps 4.1-4.7) takes about 15 min. This general approach to visualizing germline chromosomes and the gonad can be used for cytological studies of any protein if an antibody or fluorescent tag reagent exists. In its simplest form, DAPI staining of diakinesis nuclei can be used to screen for factors affecting meiotic recombination. When paired with organismal analyses of progeny number, the incidence of males (Him phenotype), and embryonic lethality, this cytological approach provides a single-cell counterpoint to population-based analyses.

Meiosis is the specialized cell division used to create gametes (eggs and sperm/pollen) in all sexually-reproducing organisms1,2. Crossover recombination is the reciprocal exchange of DNA between homologous chromosomes; it is essential for meiosis, both providing an important source of genetic diversity and promoting genome stability through generations. Chromosomes that fail to form at least one crossover during meiosis will segregate randomly, which can result in chromosome nondisjunction, creating gametes with the incorrect number of chromosomes &#8211; a condition that is usually fatal for resulting progeny3. During meiosis, crossovers are induced by programmed double-strand DNA breaks4. A subset of these breaks will be repaired as crossovers that provide physical linkages of DNA, called chiasmata, that help orient homologous chromosomes in preparation for cell division5. Meiotic stages are highly conserved across all eukaryotes, and their chromosomal conformation allows them to be easily identified.
As a fundamental concept in biology, meiosis is a topic that students encounter multiple times in different biology courses. They are often introduced to the mechanics of meiotic chromosome segregation in high school, while college-level courses focus on the cell biology of segregation and the genetic impact of crossover recombination. However, meiosis is a notoriously tricky concept for many students1. A failure to understand the relationship between genes, DNA, chromosomes, and meiosis can generate student misconceptions and gaps in understanding that impede a full understanding of genetic inheritance6,7. One way to improve student understanding of abstract topics is to provide concrete, hands-on activities. For example, when teaching meiosis, instructors can choose from activities that emulate molecular analysis8, 3D models that allow students to manipulate molecules9, or role-play where students themselves act out the molecular choreography1. Incorporating research with unknown outcomes is a particularly effective way of improving student understanding. This practice is known as a course-based undergraduate research experience (CURE) and has the added benefit of strengthening student attitudes and agency, especially for those belonging to groups that remain underrepresented in STEM10,11. The nematode worm Caenorhabditis elegans is particularly amenable for classroom studies of behavior, fertility, and genetic crosses, and is an effective model to introduce students to biological research12.
C. elegans makes a powerful model organism for cell biology by combining molecular genetics with simple cytological analysis. It is also particularly well-suited for use in a biology classroom 13,14,15. They are easy and economical to maintain in a lab, producing hundreds of progeny every 3 days, both at standard room temperature or at 20 &#176;C, the most common incubation temperature. Importantly, they can be frozen as glycerol stocks and kept in a -80 &#176;C freezer, which means that any husbandry mistakes made by novice researchers can be easily corrected16. Furthermore, its well-annotated genome allows for forward and reverse genetic techniques17,18, allowing C. elegans to be used to address biological questions ranging from the molecular to evolutionary. Finally, C. elegans researchers have created a supportive community that is often willing to provide help and advice for budding scientists19. These advantages have led to C. elegans being incorporated into a number of CUREs at various types of institutions12,19,20,21,22,23.
In addition to its benefits for research and teaching, C. elegans has become a popular model for studies of meiosis and germline development24,25,26. The optical clarity of these animals simplifies cytological approaches27, and in adults, gonads represent nearly half of the animal, providing hundreds of meiotic cells to study. In gonads, meiotic germline nuclei are arranged like an assembly line (Figure 1); mitotic replication occurs at the distal tip of the gonad, with nuclei progressing through meiotic stages as they migrate toward the proximal end of the gonad, where fertilized embryos emerge from the vulva. Because the stereotyped spatial organization also represents a temporal progression through meiosis, different stages can be easily identified based on their chromosomal organization and location in the gonad. Finally, processes that disrupt meiosis and cause aneuploidy create phenotypes that are straightforward to characterize, even for novices: sterility, embryonic lethality, or a high incidence of males (Him phenotype)28.
This is a simple protocol for visualizing meiotic chromosomes in C. elegans. Mounting, dissection, fixing, and antibody staining are all performed on the same microscope slide, which simplifies the protocol and allows near-perfect sample recovery. This method works for simple DAPI staining to visualize chromosomes and can be used for immunofluorescence to visualize the localization of proteins in the gonad. Students dissect gonads using basic dissecting microscopes, generate whole-mount preparations for visualization of DNA or immunofluorescence, and image them on a compound fluorescent microscope. This protocol has been taught to high-school students and undergraduates working in a C. elegans research lab and incorporated into a CURE at a liberal arts college12. Although the CURE had a relatively small class size, this protocol would be amenable for classes at a range of institutions due to the relatively low cost of worm strains and reagents. Instructors would only be limited by the number of dissecting microscopes available for use. The previous implementation had students working in groups of three to share a single microscope and took place over three 90-minute sessions: the first to practice dissection, the second to implement dissection and DAPI staining, and the third to image slides on a widefield fluorescence microscope. Participation in undergraduate research provides many benefits for students11,29, both academic and personal. Embedding research in courses via CUREs allows students to participate in research during normal class time11,30,31, which makes exposure to these benefits more accessible and equitable.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Rabbit IgG Secondary Antibody</td>
			<td>Molecular Probes</td>
			<td>711-545-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum heating/cooling block</td>
			<td>Millipore Sigma</td>
			<td>Z743497</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>Gibco</td>
			<td>DF0118 17 0</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Pasteur Pipets, Disposable, 5.75 inches</td>
			<td>Fisherbrand</td>
			<td>13 678 20B</td>
			<td>Used to make worm picks</td>
		</tr>
		<tr>
			<td>BSA, Bovine Serum Albumin</td>
			<td>VWR</td>
			<td>97061 420</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans wild type (ancestral)</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N2 (ancestral)</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans kle-2 (ok1151) mutants</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>VC768</td>
			<td>The full genotype of this strain is: kle-2(ok1151) III/hT2 [bli-4(e937) let-?(q782) qIs48] (I;III). It has kle-2 balanced with a balancer chromosome marked with pharangeal GFP. Pick non-green animals to identify homozygous kle-2 mutants.</td>
		</tr>
		<tr>
			<td>C. elegans spo-11 (me44) mutants</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>TY4342</td>
			<td>The full genotype of this strains is: spo-11(me44)&#160; IV / nT1[qIs51] (IV:V). It has spo-11 balanced with a balancer chromosome marked with pharangeal GFP. Pick non-green animals to identify homozygous spo-11 mutants.</td>
		</tr>
		<tr>
			<td>Calcium Chloride Anhydrous</td>
			<td>VWR</td>
			<td>97062 590</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma Aldrich</td>
			<td>501848300</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, Disodium Dihydrate Salt</td>
			<td>Apex Bioresearch Products</td>
			<td>20 147</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo Dish, glass, 30mm cavity</td>
			<td>Electron Microscopy Services</td>
			<td>100492-980</td>
			<td>Used to practice dissecting; glass is preferred because dissected animals can stick to plastic</td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>OP50</td>
			<td></td>
		</tr>
		<tr>
			<td>Food storage container, plastic</td>
			<td>various</td>
			<td>n/a</td>
			<td>Plastic containers with (1) relatively flat bottoms, to allow slides to rest level; (2) lids that are air-tight, but can still be easily removed without disturbing contents of container, and (3) are about 9 inches long by 6 inches wide are preferred</td>
		</tr>
		<tr>
			<td>Glass Coplin Jar</td>
			<td>DWK Life Sciences&#160;Wheaton</td>
			<td>08-813E</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic Barrier PAP Pen</td>
			<td>ImmEdge</td>
			<td>NC9545623</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech Science</td>
			<td>06 666A</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole Hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>L0380000</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Anhydrous</td>
			<td>Fisher Chemical</td>
			<td>M65 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles, Single-use, 25 G</td>
			<td>BD PrecisionGlide</td>
			<td>14 821 13D</td>
			<td>blue, 1 inch</td>
		</tr>
		<tr>
			<td>NGM Agar, Granulated</td>
			<td>Apex Bioresearch Products</td>
			<td>20 249NGM</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>16 101</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% w/v) Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>50 980 487</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, Phosphate Buffered Saline (10x Solution)</td>
			<td>Fisher BioReagents</td>
			<td>BP399500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes, 60-mm</td>
			<td>Tritech Research</td>
			<td>NC9321999</td>
			<td>Non-vented, sharp edge</td>
		</tr>
		<tr>
			<td>Platinum wire (90% platinum, 10% iridium)</td>
			<td>Tritech Research</td>
			<td>PT 9010</td>
			<td>Used to make worm picks</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>VWR BDH Chemicals</td>
			<td>BDH9266 500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>VWR BDH Chemicals</td>
			<td>BDH9268 500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Cover Glasses 18 mm x 18 mm</td>
			<td>Fisherbrand</td>
			<td>12-548-AP</td>
			<td>0.13&#8211;0.17 mm thickness</td>
		</tr>
		<tr>
			<td>Razor Blades, Single Edge</td>
			<td>VWR</td>
			<td>55411 055</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFade Gold Antifade Mountant</td>
			<td>Molecular Probes</td>
			<td>S36937</td>
			<td>Alternatives: VectaShield Antifade Mounting Medium (Vector Laboratories, H-1000-10) or ProLong Diamond Antifade (Thermo Fisher Scientific, P36970). If using ProLong Diamond, no nail polish is required for sealing, but slides must cure for 24 hours before imaging.</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher BioReagents</td>
			<td>BP922I 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Chemical</td>
			<td>S271 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>7558 79 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Heptahydrate</td>
			<td>Sigma Aldrich</td>
			<td>S9390</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam box</td>
			<td>various</td>
			<td>n/a</td>
			<td>Approximate dimensions of interior: 12 inches long, 9 inches wide, 8 inches deep</td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>22 037 246</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher BioReagents</td>
			<td>BP151 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Apex Bioresearch Products</td>
			<td>20 251</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Chemical</td>
			<td>BP337 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Apex Bioresearch Products</td>
			<td>20 254</td>
			<td></td>
		</tr>
	</tbody>
</table>

c. elegans gonad dissection and freeze crack for immunofluorescence and dapi staining

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.

DAPI binds strongly to DNA, and its staining is robust even under a wide variety of conditions (Figure 3A,B). It should be present in all nuclei and therefore makes an effective positive control for the presence of any worm tissue on the slide and the ability to detect fluorescence on the microscope. Staining is effective when the antibody is present within meiotic nuclei (Figure 3A,B). For example, Figure 3A,B shows mid-pachytene nuclei stained with DAPI and an antibody targeting RAD-51, a marker for double-strand breaks. KLE-2 is a component of condensin, a highly conserved protein complex that structures chromosomes in preparation for mitosis and meiosis. kle-2/+ mutants have minor defects in chromosome structure, as shown by slightly disordered DAPI staining and an increase in double-strand break number reflected in the higher number of RAD-51 foci (Figure 3B). A common mistake for immunofluorescence is leaving the sample in the fix solution for too long. Over-fixing can lead to unsuccessful staining because it usually prevents antibodies from diffusing into nuclei. This mistake can be identified when the antibody is diffused in the cytoplasmic regions of the gonad, but appears to be excluded from nuclei.
In the germline (Figure 1), nuclei mitotically proliferate at the distal tip, enter meiosis during the transition zone, and progress through pachytene (which is often divided into three equal stages by a number of nuclei rows) before entering diplotene and finally diakinesis. Oocytes in diakinesis are numbered based on their proximity to the spermatheca, with the -1 oocyte being most proximal to the spermatheca, the -2 oocyte being the next distal, and so on. C. elegans have six chromosomes, which manifest as compact ovals in diakinesis nuclei. Each of these represents a homologous pair of two sister chromatids held together by a chiasma, also called a bivalent (Figure 3C). Mutants, like spo-11, that disrupt crossover formation will fail to form chiasmata; therefore, diakinesis nuclei will have 12 separate DAPI bodies (also called univalents), one for each sister chromatid (Figure 3D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64204/64204fig01.jpg" /> 
Figure 1: Germline nuclei are arrayed in a spatial-temporal manner that represents their progression through meiosis. (A)&#160;Whole-mount young adult hermaphrodite stained with DAPI. Gonad and meiotic stages are outlined, from the distal tip (asterisk) to the proximal region (the -1 oocyte, indicated with an arrow), which contains the spermatheca (triangle). Scale bar represents 100 &#181;m. (B)&#160;Projected fluorescence image of a dissected hermaphrodite gonad stained with DAPI. Meiotic stages are denoted, with representative nuclei shown in insets (all insets are shown to scale with one another). Nuclei can be easily staged based on their location in the germline and characteristic chromosome morphology, progressing from the mitotic zone at the distal tip (asterisk), to the transition zone, through pachytene, diplotene, and diakinesis. The spermatheca is marked by a triangle. This figure has been adapted from Hillers et al. under license CC BY 3.028. <a href="https://www.jove.com/files/ftp_upload/64204/64204fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64204/64204fig02.jpg" /> 
Figure 2: Demonstration of dissection setup. (A) Microscope stage during dissection. Animals are dissected in 4 &#181;L of dissection solution on a coverslip placed on the holding slide, which is used to move the coverslip into view. The diagram shows ideal needle placement. Needles are held with a beveled edge facing down, crossed over the pharyngeal region. Pink arrows demonstrate a scissor-like motion for needles. The pink dashed line shows the ideal cut location. (B) Images of dissected animals, with an example of a full extrusion and an incomplete extrusion. Sections of extruded gonads and guts are labeled. (C) Image demonstrating the freeze-crack step. The slide should be held firmly in one hand, with the coverslip side facing away from the body. The opposite edge is braced against the bench. The razor should be held in the other hand. The pink arrow indicates the direction of movement for the razor to flick the coverslip off the slide, with a slight turn such that the coverslip flicks away from the body. Note that the coverslip has been placed such that one corner hangs over the long edge of the slide. (D) Image of the setup for dissection practice in a glass embryo staining dish. Animals are placed in 50-200 &#181;L of dissection solution, which negates the problem of evaporation and extends the time for dissection. <a href="https://www.jove.com/files/ftp_upload/64204/64204fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64204/64204fig03.jpg" /> 
Figure 3: Representative fluorescence results of germline nuclei. (A,B) Z-stack projections of late pachytene nuclei stained with RAD-51 antibody (green) and DAPI (red) in (A) wild-type and (B) kle-2/+ heterozygotes. (C,D) Z-stack projections of a single diakinesis nucleus stained with DAPI in (C) wild-type (with six DAPI staining bodies) and (D) spo-11 mutants (with 12 DAPI staining bodies). Scale bars represent 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64204/64204fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining at JoVE.com

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

This is a commonly used method for C. elegans gonad dissection followed by freeze crack, which produces germline samples for immunofluorescence via antibody staining, or for simple DAPI staining to visualize DNA. This protocol has been successful for undergraduates in a research lab and in a course-based undergraduate research experience.

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. ...

c-elegans-gonad-dissection-freeze-crack-for-immunofluorescence-dapi

Research

JoVE Journal

Genetics

4.9K Views.  University of Massachusetts Lowell. This is a commonly used method for C. elegans gonad dissection followed by freeze crack, which produces germline samples for immunofluorescence via antibody staining, or for simple DAPI staining to visualize DNA. This protocol has been successful for undergraduates in a research lab and in a course-based undergraduate research experience.

Video: C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing&#160;in less than two weeks.

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The goal of this protocol is to enable seamless CRISPR/Cas9 editing of the C. elegans genome using assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. This protocol can be used to make a variety of genetic modifications in C. elegans including insertions, deletions, gene replacements and codon substitutions.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been ...