Name: high-throughput dna extraction and genotyping of 3dpf zebrafish larvae by fin clipping
Uploaded: 2018-06-29T15:00:00
Description: Watch this Scientific Journal Video about High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping at JoVE.com

The authors would like to thank the Rare Disease Models and Mechanism (RDMM) Network (funded by the Canadian Institutes of Health Research (CIHR) and Genome Canada). CK is supported by a UROP scholarship award (University of Ottawa). DLJ is supported by a Vanier Canada Graduate Scholarship. IAP is supported by a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship award. The authors thank the Genetics Society of America for granting permission to publish this protocol and to use an adaptation of the Supplemental Figure 5 from Pena, et al.

Procedures involving animal subjects have been approved and are in accordance with animal care guidelines provided by the Canadian Council on Animal Care and the University of Ottawa animal care committees.
1. Preparation
<ol>
	<li>Prepare the dissection surface by taping a 9 cm Petri dish lid with autoclave tape across its interior surface. Position it under a stereo-microscope.</li>
	<li>Place 3 - 5 days post-fertilization (dpf) zebrafish larvae in a Petri dish and anesthetize in &#732;1.5 mM Tricaine in 1x E3...

<ol>
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Gene editing tools have been employed to precisely introduce mutations and transgenes in zebrafish over the past years5,6,7,8. When performing disease modeling in zebrafish, early larval or juvenile phenotypes are often observed9,12,13,14. The protocol presented here ...

The zebrafish (Danio rerio) is a vertebrate organism widely used as a model for disease investigation as well as for preclinical testing of therapeutic hypotheses1,2,3. These animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos4. Through the increasing development of novel transgenic and gene editing technologies such as Zinc-Finger nucleases (ZFN)5, TAL-like Effector Nucleases (TALENs)6,7, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system8, the zebrafish is poised to substantially improve the understanding of several pathologic conditions. These technologies have been used to create targeted knockouts in both somatic and germline cells in zebrafish, efficiently engendering genetically modified animals8. Recent examples in the literature include the successful development of genetic models of epilepsy and metabolic disorders in zebrafish3,9,10,11,12.
With the progress made in zebrafish genome editing, rapid and reliable genotyping has become an undeniable rate-limiting step. Identification of specific DNA mutations adjacent to the predicted genome-editing target site is an essential stage of the protocol. Traditionally, genotyping techniques by fin clipping are usually only performed in juvenile or adult zebrafish. However, the time spent raising zebrafish to adulthood (&#62;2 months) considerably delays advancements in research. In many cases, adulthood cannot be achieved due to the knockout of essential genes (e.g., in disease modeling) or gain-of-function mutations leading to detrimental phenotypic effects. In addition, several assays require pooling of larvae, such as in RNA or metabolite extraction, and thus involve prior identification of the genotypes, which can be challenging when only post hoc genotyping techniques are available. These aforementioned examples highlight the importance of larval genotyping techniques which are at the same time precise and enable full recovery and normal development of the larvae. Early stage zebrafish genotyping does not currently appear to be widely used; this is reflected by recent publications of disease models in which larval genotypes are identified via post-mortem genotyping rather than genotyping prior to the execution of the experiment12,13,14. In this paper, a larval fin clip strategy is presented aiming to improve previously established genotyping procedures.
In the past, strategies employing proteinase K digestion to genotype live zebrafish larvae have led to variable polymerase chain reaction (PCR) efficiency15. Although a more recently published microscopic tail biopsy technique provided more promising results16, we and other groups were not able to reproduce the high efficiency and DNA recovery reported by the authors. A major setback of the protocol described by Wilkinson&#160;et al.16 could be the transfer of the tail tissue to the PCR tube. Due to its microscopic size, it is hard to ensure that the fin was properly collected and dispensed by pipetting. To address this barrier, we have developed an improved protocol for genotyping large numbers of live zebrafish larvae with nearly 100% efficiency9. Using a microscalpel, the tip of the fin tail is removed and placed onto a piece of filter paper. The piece of filter paper containing the tissue can be readily visualized and correctly placed into a PCR tube. Genomic DNA extraction is then performed, followed by PCR amplification of the region of interest to genotype the specimen. The advantages of this method include a high PCR efficiency, a low false positive rate and a low mortality rate. Additionally, genotyping of large numbers of embryos is possible using this protocol. The protocol described herein allows fin-clipping of hundreds of larvae within 2 - 3 h using this approach and correct identification of genotyped fish and tail regeneration within two days.

<table>
	<tbody>
		<tr>
			<td>Petri dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscalpel</td>
			<td>World Precision Instruments</td>
			<td>500249</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine/Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Tissue-culture plate</td>
			<td>Costar</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well PCR plate</td>
			<td>Fisher</td>
			<td>14230232</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher</td>
			<td>S25548A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Chelex 100 sodium form</td>
			<td>Sigma-Aldrich</td>
			<td>C7901</td>
			<td></td>
		</tr>
		<tr>
			<td>GelRed 1x</td>
			<td>Biotium</td>
			<td>41003</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-Cell Model 192 system</td>
			<td>Bio-Rad&#160;</td>
			<td>1704508</td>
			<td></td>
		</tr>
		<tr>
			<td>30 % Acrylamide/Bis Solution, 37.5: 1</td>
			<td>Bio-Rad&#160;</td>
			<td>1610158</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq Green Master Mix, 2X</td>
			<td>Promega</td>
			<td>M7123</td>
			<td></td>
		</tr>
		<tr>
			<td>paper wipes (Kimwipes&#174; disposable wipers)</td>
			<td>Sigma-Aldrich</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>1kb-plus molecylar weight marker&#160;</td>
			<td>Thermo Scientific</td>
			<td>SM1343</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sub-Cell Model 192 system (high-throughput agarose gel system)</td>
			<td>Bio-Rad&#160;</td>
			<td>1704508</td>
			<td></td>
		</tr>
		<tr>
			<td>vertical gel electrophoresis system Mini-PROTEAN Tetra Cell&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>1658004</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput dna extraction and genotyping of 3dpf zebrafish larvae by fin clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

The efficiency of the protocoll was demonstrated by genotyping the offspring of an heterozygous cross (aldh7a1+/ot100, here shown as aldh7a1+/-) (Figure 3A-C)9. The aldh7a1ot100 mutant allele has a 5-base pair (bp) insertion in the first coding exon of the zebrafish aldh7a1 gene that leads to frameshift and loss-of-function due to an ...

Watch this Scientific Journal Video about High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping at JoVE.com

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

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