Name: screening sperm for the rapid isolation of germline edits in zebrafish
Uploaded: 2023-02-10T12:10:00
Description: Watch this Scientific Journal Video about Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish at JoVE.com

We would like to thank Anna Hindes at Washington University School of Medicine for her initial efforts in obtaining good-quality sperm genomic DNA using the hot shot method. This work was funded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award (R01AR072009 to R.S.G.).

This study was carried out in line with the guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Texas at Austin Animal Care and Use Committee (AUP-2021-00254).
1. Designing the sgRNA for CRISPR mutagenesis
<ol>
	<li>Obtain the exon sequence containing the targeted loci.</li>
	<li>Design a synthetic guide RNA (sgRNA) with a protospacer adjacent motif (PAM) site that is specific...

<ol>
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R., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+multiplex+biallelic+zebrafish+genome+editing+using+a+CRISPR+nuclease+system.">Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (34), 13904-13909 (2013).</li><li>Troutwine, B. 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=The+Reissner+fiber+is+highly+dynamic+in+vivo+and+controls+morphogenesis+of+the+spine.">The Reissner fiber is highly dynamic in vivo and controls morphogenesis of the spine.</a> Current Biology. 30 (12), 2353-2362 (2020).</li><li>Xu, Y., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems:+Overview,+innovations+and+applications+in+human+disease+research+and+gene+therapy.">CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy.</a> Computational and Structural Biotechnology Journal. 18, 2401-2415 (2020).</li><li>Creaser, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technic+of+handling+the+zebrafish+(Brachydanio+rerio)+for+the+production+of+eggs+which+are+favorable+for+embryological+research+and+are+available+at+any+specified+time+throughout+the+year.">The technic of handling the zebrafish (Brachydanio rerio) for the production of eggs which are favorable for embryological research and are available at any specified time throughout the year.</a> Copeia. 1930 (4), 159-161 (1934).</li><li>Carmichael, C., Westerfiel, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservatin+and+in+vitro+fertilization+at+the+zebrafish+international+resource+center.">Cryopreservatin and in vitro fertilization at the zebrafish international resource center.</a> Methods in Molecular Biology. 546, 45-65 (2009).</li><li>Wang, Y., Troutwine, B. R., Zhang, H., Gray, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axonemal+dynein+heavy+chain+10+gene+is+essential+for+monocilia+motility+and+spine+alignment+in+zebrafish.">The axonemal dynein heavy chain 10 gene is essential for monocilia motility and spine alignment in zebrafish.</a> Developmental Biology. 482, 82-90 (2021).</li><li>Gray, R. S., 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=Postembryonic+screen+for+mutations+affecting+spine+development+in+zebrafish.">Postembryonic screen for mutations affecting spine development in zebrafish.</a> Developmental Biology. 471, 18-33 (2021).</li><li>Brocal, I., 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=Efficient+identification+of+CRISPR/Cas9-induced+insertions/deletions+by+direct+germline+screening+in+zebrafish.">Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish.</a> BMC Genomics. 17, 259 (2016).</li><li>Davis, M. W., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ApE,+A+Plasmid+Editor:+A+freely+available+DNA+manipulation+and+visualization+program.">ApE, A Plasmid Editor: A freely available DNA manipulation and visualization program.</a> Frontiers in Bioinformatics. 2, 816619 (2022).</li><li>Labun, K., 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=CHOPCHOP+v3:+Expanding+the+CRISPR+web+toolbox+beyond+genome+editing.">CHOPCHOP v3: Expanding the CRISPR web toolbox beyond genome editing.</a> Nucleic Acids Research. 47, 171-174 (2019).</li><li>Hill, J. T., 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=Poly+Peak+Parser:+Method+and+software+for+identification+of+unknown+indels+using+sanger+sequencing+of+polymerase+chain+reaction+products.">Poly Peak Parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products.</a> Developmental Dynamics. 243 (12), 1632-1636 (2014).</li><li>Varshney, G. K., 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=High-throughput+gene+targeting+and+phenotyping+in+zebrafish+using+CRISPR/Cas9.">High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.</a> Genome Research. 25 (7), 1030-1042 (2015).</li><li>Liu, Q., 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=Hi-TOM:+A+platform+for+high-throughput+tracking+of+mutations+induced+by+CRISPR/Cas+systems.">Hi-TOM: A platform for high-throughput tracking of mutations induced by CRISPR/Cas systems.</a> Science China. Life Sciences. 62 (1), 1-7 (2019).</li><li>Sorlien, E. L., Witucki, M. A., Ogas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+production+and+identification+of+CRISPR/Cas9-generated+gene+knockouts+in+the+model+system+Danio+rerio.">Efficient production and identification of CRISPR/Cas9-generated gene knockouts in the model system Danio rerio.</a> Journal of Visualized Experiments. (138), e56969 (2018).</li><li>Draper, B. W., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+zebrafish+sperm+cryopreservation+and+in+vitro+fertilization.">A high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization.</a> Journal of Visualized Experiments. (29), e1395 (2009).</li><li>Parant, J. M., George, S. A., Pryor, R., Wittwer, C. T., Yost, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+of+genotyping+zebrafish+mutants.">A rapid and efficient method of genotyping zebrafish mutants.</a> Developmental Dynamics. 238 (12), 3168-3174 (2009).</li></ol>

This protocol describes a method for rapidly characterizing putative genome edits or targeted mutations using CRISPR-Cas technology by focused analysis on F0 male sperm genomes. This protocol should be amenable to other animal models where sperm is readily available for sampling without euthanasia. This method will increase the throughput of screening for desired edits and is especially useful for identifying rare HDR-mediated knock-in events. This approach also serves to reduce the number of experimental animals used to...

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a powerful tool to perform loci-specific mutagenesis and precise genome editing in the Danio rerio (zebrafish) model system1,2,3,4. The Cas-ribonucleoprotein (RNP) is comprised of two main components: a Cas endonuclease (commonly Cas9 or Cas12a) and a locus-specific synthetic guide RNA (sgRNA)5. Together, the Cas-RNP generates a double-stranded break (DSB) in the desired locus that can be repaired by one of two intrinsic repair mechanisms. The non-homologous end joining (NHEJ) repair mechanism is error-prone and often results in a variety of insertions or deletions (indels) around the DSB. These indels can be deleterious if they introduce a frameshift mutation or premature stop in the resultant protein sequence. Alternatively, the homology-directed repair (HDR) mechanism uses a donor template with regions of homology surrounding the DSB site to repair the damage. Researchers can take advantage of the HDR system to generate precise genomic edits. Specifically, they can co-inject a double-stranded DNA donor construct that contains the desired edits as well as regions of homology flanking the DSB site in the genome. The increased economy of scale for these commercially produced CRISPR components has greatly reduced the barriers to screening multiple loci and to setting up larger-scale efforts for precise genome editing. However, in sexually reproducing animal models, a major bottleneck is the identification and isolation of germline-stable mutant animals.
The zebrafish model system exhibits several key qualities that enhance its use in reverse genetic studies. They are easy to rear in large numbers with basic aquatic housing equipment, and females exhibit high fecundity all year round6. Moreover, their external egg-laying and fertilization make them amenable to the microinjection of CRISPR/Cas components. The Cas-RNP is commonly injected into one-cell stage zebrafish embryos to generate DSBs/repair that is, in theory, inherited by all the daughter cells. However, diploid genomes require two DSB/repair events to mutagenize both homologous chromosomes. Furthermore, although Cas-RNP is injected at the one-cell stage, the DSB/repair may not occur until later points in development. Together, these factors contribute to the mosaic nature of F0-injected fish. A common practice is to outcross F0-injected fish and screen the F1 progeny for indels/specific edits. However, since not all F0-injected fish possess germline mutations, this practice results in many unproductive crosses that do not generate the desired edit. Screening the F0 germline rather than F1 somatic tissue increases the probability of isolating the desired germline edit and reduces the number of animals required in this process.
Sperm can easily be collected from F0-injected zebrafish without the need for euthanasia. This feature allows for the cryopreservation and rederivation of frozen sperm stocks7 but can also be exploited to rapidly screen, identify, and isolate the germline carriers of desired genomic mutations8,9. Brocal et al. (2016) previously described a sequencing-based method for screening germline edits in F0-injected male zebrafish10. Although useful for identifying the mutated alleles present in the germline, this approach can become costly in high throughput and may not be accessible for all labs. In contrast, the current protocol offers an approachable and cost-effective electrophoresis-based strategy for identifying germline edits. Specifically, this protocol outlines a robust method for screening and isolating germline mutations at specific loci using high-resolution agarose gel electrophoresis. In addition, this protocol describes a similar strategy for identifying the successful integration of a donor construct containing specific edits. As always, if specific edits are desired, sequencing-based strategies can be performed in tandem with the protocol described below. Although this protocol is specific to the zebrafish model system, these principles should be adaptable to any model in which the collection of sperm is a routine procedure. Together, these strategies will allow for the identification of F0-injected males with germline indels/edits&#160;that can be resolved on a gel after standard polymerase chain reaction (PCR) and/or restriction digest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose powder&#160;</td>
			<td>Fisher BioReagents</td>
			<td>BP1356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Breeding tanks</td>
			<td>Carolina Biological&#160;</td>
			<td>161937</td>
			<td></td>
		</tr>
		<tr>
			<td>BstNI Restriction Enzyme</td>
			<td>NEB</td>
			<td>R0168S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 Endonuclease</td>
			<td>IDT</td>
			<td>1081060</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Ladder, 100 bp&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>FERSM0241</td>
			<td></td>
		</tr>
		<tr>
			<td>dnah10 donor construct&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry. 
			Phosphorothioate bond on the donor at the first three phosphate bonds on both the 5&#8217; and 3&#8217; ends (5&#39;-CCTCTCTCCCTTTCAGAAGCTTC 
			TGCTCATCCGCTGCTTCTGCCT 
			GGACCGAGTGTACCGTGCCGTC 
			AGTGATTACGTCACGC-3&#39;)</td>
			<td></td>
		</tr>
		<tr>
			<td>dnah10 forward primer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (5&#39;-CATGGAACTCTTTCCTAATGAGT 
			TTGGC-3&#39;)</td>
			<td></td>
		</tr>
		<tr>
			<td>dnah10 reverse primer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (&#39;5-AGTAGAGATCACACATCAACAGA 
			ATACAGC-3&#39;)&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>dnah10 synthetic sgRNA&#160;</td>
			<td>Synthego&#160;</td>
			<td>Synthetic sgRNA, target sequence: 5&#39;-GCTCATCCGCTGCTTCAGGC-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis power supply</td>
			<td>Thermo Scientific&#160;</td>
			<td>105ECA-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter forceps</td>
			<td>Millipore</td>
			<td>XX6200006P</td>
			<td></td>
		</tr>
		<tr>
			<td>Fish (system) water</td>
			<td>Generic</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel electrophoresis system (including casting frame, comb, and electrophoresis chamber)&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>B2</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel imaging light box&#160;</td>
			<td>Azure Biosystems</td>
			<td>AZI200-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel stain, 10000X&#160;</td>
			<td>Invitrogen</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bowl, 250 mL&#160;</td>
			<td>Generic</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolation tanks, 0.8 L&#160;</td>
			<td>Aquaneering</td>
			<td>ZT080</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcap capillary tube with bulb, 20 &#181;L&#160;</td>
			<td>Drummond</td>
			<td>1-000-0020/CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Minicentrifuge</td>
			<td>Bio-Rad</td>
			<td>12011919EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes, various with appropriate tips</td>
			<td>Generic&#160;</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave&#160;</td>
			<td>Generic&#160;</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Promega</td>
			<td>P119-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>Generic</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes, 0.2 mL</td>
			<td>Bioexpress</td>
			<td>T-3196-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic spoon, with drilled holes/slots&#160;</td>
			<td>Generic</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl solution, 0.2 M RNAse Free</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>p2ry12 forward primer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (5&#39;-CCCAAATGTAATCCTGACCAGT 
			-3&#39;)</td>
			<td></td>
		</tr>
		<tr>
			<td>p2ry12 reverse primer</td>
			<td>Sigma-Aldrich</td>
			<td>DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (5&#39;-CCAGGAACACATTAACCTGGAT 
			-3&#39;)&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>p2ry12 synthetic sgRNA&#160;</td>
			<td>Synthego&#160;</td>
			<td>Synthetic sgRNA, target sequence: 5&#39;-GGCCGCACGAGGTCTCCGCG-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction Enzyme 10X Buffer</td>
			<td>NEB</td>
			<td>B6003SVIAL</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH solution, 50 mM&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>S318; 424330010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sponge, 1-inch x 1-inch cut with small oval divot</td>
			<td>Generic&#160;</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss</td>
			<td>Stemi 508</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq polymerase master mix, 2X</td>
			<td>Promega</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>TBE Buffer Concentrate, 10X</td>
			<td>VWR</td>
			<td>E442</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue paper</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine-methanesulfonate solution (Syncaine, MS-222), 0.016% in fish water (pH 7.0&#177;0.2)&#160;</td>
			<td>Syndel</td>
			<td>200-266</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base, 1M (Buffered with HCl to ph 8.0)&#160;</td>
			<td>Promega</td>
			<td>H5131</td>
			<td></td>
		</tr>
	</tbody>
</table>

screening sperm for the rapid isolation of germline edits in zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

The experimental approaches described in this protocol allow for the more rapid identification of genome edits or putative deleterious alleles by focusing on the analysis of thousands of genomes derived from the collection of F0-injected male sperm. Figure 2 highlights how to interpret the results obtained using this protocol.
To generate mutations in the p2ry12 locus, one-cell stage zebrafish embryos were injected with Cas9 endonuclease and a p2ry12<...

Watch this Scientific Journal Video about Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish at JoVE.com

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

This protocol describes a method for quickly screening thousands of genomes using F0 CRISPR-injected zebrafish sperm. This technique is advantageous in its accessibility. Rather than expensive, specialized sequencing-based approaches, our method uses PCR restriction enzymes and gel electrophoresis to screen F0 male carriers for indels or specific genome edits.The hardest part of this procedure is just getting the sperm quickly, but it becomes a good technique for in vitro fertilization if we work with a lot of dysmorphic fish, and so it's an easy way for us to cross fish that are giving us trouble. Begin by setting up breeding tanks for both the wild type and F0 fish and incubate the tanks overnight. After anesthetizing the male fish the next day, use a slotted spoon to transfer it onto a stack of clean paper towels.Gently roll the fish to remove excess water. Remove any water by blotting it dry with a clean folded tissue. Using the slotted spoon, transfer the fish to the prepared sponge.Place the fish ventral-side up and gently blot the area around the anal fins with a clean folded tissue paper. To collect the sperm, use a capillary tube to gently move the pelvic fins away from the midline, exposing the cloaca. Place the capillary tube near the cloaca.With your fingers or a pair of filter forceps, gently squeeze the sides of the fish from the gills down. Using capillary action, collect the sperm in the tube and place it in a PCR tube containing 40 microliters of 50 millimolar sodium hydroxide solution, then expel the sample into the tube. After spinning the sperm samples in a mini centrifuge, place the PCR tubes into a thermal cycler.Run the cycler by heating the samples for 40 minutes at 95 degrees Celsius followed by cooling at 25 degrees Celsius. After removing the samples from the cycler, neutralize the pH by adding 10 microliters of one molar tris hydrochloride of pH eight. Mix by aspirating the suspension and then centrifuge the samples.Set the thermal cycler to 95 degrees Celsius for preheating. And in the meantime, prepare 25 microliters of the PCR reaction mixture for each sperm sample. Mix well by pipetting up and down, then spin the samples.Place the samples in the preheated thermal cycler and set the cycle. To obtain a gel solution, microwave a 4%agarose gel solution prepared by mixing agarose powder, gel stain, and TBE buffer. Pour the gel solution into a gel casting frame and insert a comb on one side.After gel solidification, carefully remove the comb. Place the gel in an electrophoresis rig such that the wells are closest to the negative electrode. Pour the TBE running buffer into the rig until the wells are fully submerged.Begin to load the gel by pipetting five microliters of DNA ladder into the first well. Load the remaining wells with 5 to 10 microliters of the PCR product. Run the gel for 1 hour at 150 volts to ensure adequate separation of the amplicons.Use the gel imager to view the DNA bands. p2ry12 locus analysis showed that the wild-type amplicon displayed a single band of 250 base pair length while the F0-injected male amplicons containing indels ran as multiple bands. dnah10 locus analysis showed that wild-type amplicon run as a single 400 base pair long band.F0-injected male amplicons containing indels ran as multiple bands or as a single band with decreased gel mobility. The F0-injected male number one had an additional band in the digested product, which was absent in the PCR product, rendering it the best founder candidate for mutant knock-in line establishment. If a male fish does not produce sperm when squeezed, it is best to rest the fish for a week and try again instead of squeezing them too hard and risk hurting them.Once the FC or male founder is outcross, the alleles need to be sequence verified in the F1 progeny. Sequencing the F1 amplicons directly and performing heterozygous allele analysis is an easy way to do this.

Research

JoVE Journal

Genetics

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