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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A., 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=Pyridoxine-Dependent+Epilepsy+in+Zebrafish+Caused+by+Aldh7a1+Deficiency.">Pyridoxine-Dependent Epilepsy in Zebrafish Caused by Aldh7a1 Deficiency.</a> Genetics. 207, 1501-1518 (2017).</li><li>Baraban, S. C., Dinday, M. T., Hortopan, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+in+Scn1a+zebrafish+mutant+identifies+clemizole+as+a+potential+Dravet+syndrome+treatment.">Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment.</a> Nat Commun. 4, 2410 (2013).</li><li>Godoy, R., Noble, S., Yoon, K., Anisman, H., Ekker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemogenetic+ablation+of+dopaminergic+neurons+leads+to+transient+locomotor+impairments+in+zebrafish+larvae.">Chemogenetic ablation of dopaminergic neurons leads to transient locomotor impairments in zebrafish larvae.</a> J Neurochem. 135, 249-260 (2015).</li><li>Scheldeman, 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=Neurobiology+of+Disease+mTOR-related+neuropathology+in+mutant+tsc2+zebra+fish+Phenotypic,+transcriptomic+and+pharmacological+analysis.">Neurobiology of Disease mTOR-related neuropathology in mutant tsc2 zebra fish Phenotypic, transcriptomic and pharmacological analysis.</a> Neurobiol Dis. 108 (September), 225-237 (2017).</li><li>Zabinyakov, N., 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=Characterization+of+the+first+knock-out+aldh7a1+zebrafish+model+for+pyridoxine-dependent+epilepsy+using+CRISPR-Cas9+technology.">Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.</a> PLoS One. 12 (10), 1-14 (2017).</li><li>Grone, B. 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=Epilepsy,+behavioral+abnormalities,+and+physiological+comorbidities+in+syntaxin-binding+protein+1+(STXBP1)+mutant+zebrafish.">Epilepsy, behavioral abnormalities, and physiological comorbidities in syntaxin-binding protein 1 (STXBP1) mutant zebrafish.</a> PLoS One. 11 (3), (2016).</li><li>Kawakami, K., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+transgenic+zebrafish.">Rapid identification of transgenic zebrafish.</a> Trends Genet. 12 (1), 9-10 (1996).</li><li>Wilkinson, R. N., Elworthy, S., Ingham, P. W., van Eeden, F. J. 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A., Higuchi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chelex+100+as+a+medium+for+simple+extraction+of+DNA+for+PCR-based+typing+from+forensic+material.">Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material.</a> Biotechniques. 54 (3), 506-513 (2013).</li><li>Ewing, B., Green, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basecalling+of+automated+sequencer+traces+using+phred.+II.+Error+probabilities.">Basecalling of automated sequencer traces using phred. II. Error probabilities.</a> Genome Res. 8, 175-185 (1998).</li></ol>

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 ...

This method can help answer key questions in the biomedical field. Such as the use of zebrafish to generate genetic models of disease. The main advantage of this technique is that it allows early identification of genotypes using a simple high-throughput method.Though this method can enable extensive research using zebrafish models of genetic epilepsies, it can also be applied to other genetic disease models and any type of mutation study. Generally, individuals new to this method may have difficulties as the transection of the larval fin must be precisely performed within the pigment gap of the tail. Prior to starting the fin clipping of the zebrafish larvae, prepare the dissection surface.Tape a piece of autoclave tape across the interior surface of a nine centimeter Petri dish lid and position the lid under a stereo microscope. Prepare a P1000 pipette tip for accommodating a three dpf larva with minimal stress, by cutting off the end of the pipette tip to a diameter of two millimeters. Place three to five days post fertilization, or dpf, zebrafish larvae in a Petri dish and anesthetize in 1.5 millimolar tricaine in 1x E3 embryo medium.Using the modified P1000 micro pipette, pick up and anesthetize zebrafish larva and place it onto the positioned Autoclave tape on the Petri dish lid. Remove excess tricaine solution surrounding the larva. The area should be as dry as possible to successfully section and pick up the fin, but still wet to ensure survival.To avoid bleeding and ensure survival of the larva during fin clipping, the transection must occur within the pigment gap. Distal to the limit of the blood circulation, as indicated in this image. Working under a stereo microscope, use a micro scalpel to section the caudal fin.During the incision, apply a steady but gentle downward pressure to avoid damaging the notochord. Visualize the sectioned fin under the microscope, and position the piece on top of the tip of the micro scalpel blade. Place the micro scalpel containing the fin onto the surface of a small piece of filter paper.Due to the presence of melanocytes in the transected tissue, the fin appears as a small black spot. To ensure successful DNA extraction, it is important that a black dot marking the placement of the fin is observed on the filter paper before continuing with the protocol. Using scissors and tweezers, transfer the filter paper containing the fin to a well of a 96-well PCR plate containing 25 microliters of a 50 millimolar sodium hydroxide solution.Next, prepare a flat bottom 96-well tissue culture plate, numbering the wells according to the label of the PCR plate. Using the modified P1000 pipette, filled with 200 microliters of fresh e3 embryo medium, carefully collect the zebrafish larva using gentle pressure. Dispense the larva in the 96-well plate in the same well number as the PCR plate.Make sure the filter paper containing the fin is well submerged in the solution. To prevent cross contamination between each cut, clean the blade of the micro scalpel and the tweezers by dipping into a 70%ethanol solution. Wipe the blade with a clean paper tissue or wipe.When all larvae have been clipped, store the larvae in the 96-well plate at 28 degrees Celsius until their genotypes have been identified. Begin this procedure by sealing the 96-well PCR plate containing the fin pieces and centrifuging the samples at 1000 times G for one minute. To make sure all the filter papers are submerged in the sodium hydroxide solution.For tissue lysis, heat the samples in a thermal cycler at 95 degrees Celsius for five minutes. Followed by cooling to four degrees Celsius for 10 minutes. Next, add six microliters of 500 millimolar tris-HCL pH8.0 to each sample.Vortex the samples. Briefly centrifuge the plate at 1500 times G for five minutes at room temperature. To extract genomic DNA using this method, clip the fin as demonstrated earlier, but add the filter paper with the clipped fin to a well of a 96-well PCR plate containing 30 microliters of 5%chelating resin.After all fin pieces have been clipped, seal the 96-well PCR plate and briefly centrifuge the samples to make sure all filter papers are submerged with the chelating resin. Heat the samples in a thermal cycler at 95 degrees Celsius for 15 minutes, followed by cooling to four degrees Celsius for 10 minutes. Briefly centrifuge the samples to pellet the resin beads and obtain the DNA in the suspension.A multiplex PCR assay will enable the discrimination between homozygous mutants, heterozygotes, and wild type larvae. Set up the PCR reaction as detailed in the text protocol. Perform the PCR reactions in 96-well thermal cycler.Prepare a 1%agarose gel in sodium borate buffer, stained with 1x gel red. To facilitate the process, if possible, use a gel system that is compatible with multi channel pipettes to enable higher throughput capabilities by reducing the time to load samples. Run the gel in 1x sodium borate buffer at 250 volts for 15 minutes.Image the gel under ultraviolet light to allow discrimination of the different genotypes. Start this assay by preparing two 12%polyacrylamide gels using the 1.5 millimeter spacer plate and the 15 sample combs as described in the text protocol. Use a vertical electrophoresis system and 1x TBE buffer as the running buffer.After performing PCR as described in the text protocol, heat the PCR products at 94 degrees Celsius for five minutes, followed by cooling at four degrees Celsius for 10 minutes. Load 10 microliters per PCR sample, and eight microliters of molecular weight marker. Run the gel at 150 volts for one hour, or until the molecular weight marker almost reaches the foot line of the glass plate.Image the gel under ultraviolet light to allow discrimination of the different genotypes. This protocol was used to genotype of the offspring of a heterozygous cross, in which the mutant ileal has a 5 base pair insertion in the first coating exon of the aldh7a1 gene. The expected result from PCR amplification of both wild type and mutant ileals is a 434 BP amplicon.A 293 BP band arises from amplification of the mutant ileal, and a 195 BP band is obtained for the wild type ileal. Analysis of PCR reactions on a 1%agarose sodium borate gel identified wild type genotypes in lanes two and six, heterozygous genotypes in lanes three, four, and five, and a homozygous mutant genotype in lane one. Various mutations where genotyped by a heteroduplex melting assay.The plpbpot101 and plpbpot102 mutant ileals of the plpbp gene each let to a frame shift and early stop codon. After denaturation and annealing, the PCR fragments for heterozygous animals would contain heteroduplex and homoduplex DNA that are easily separated and visualized using polyacrylamide gel electrophoreses. Compound heterozygous mutant genotypes were identified in lanes two, four, and eight, heterozygous genotypes in lanes three, six, seven, nine, and ten, and a wild type genotype in lane five.Once mastered, fin clipping 96 larvae using this technique can be done in less than an hour, if performed properly. Following this procedure, other methods, like pooling of zebrafish larvae of the same genotype for tissue extraction, can be performed and ordered to answer additional questions, such as, biomarker investigation and disease models. After it's development, this technique paved the way for research in the field of models of metabolic epilepsy to explore disease path physiology in zebrafish.After watching this video, you should have a good understanding of how to properly genotype three day old zebrafish larvae by microdissection of a caudal fin.

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