Name: Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting (Video) | JoVE Journal
Uploaded: 2020-01-29T13:00:00.000+00:00
Duration: 7 min 17 s
Description: Learn embryonic grafting techniques to generate chimeric axolotls with mutant haploid limbs for genetic screening. This method enables CRISPR-Cas9 mutagenesis studies and rescues embryonic lethal phenotypes.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We would like to thank Katherine Roberts for her care of the axolotl colony. Funding for this work was provided by the Connecticut Innovations Regenerative Medicine Research Fund (15RMA-YALE-09 and 15-RMB-YALE-01) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (Individual Postdoctoral Fellowship F32HD086942).

Experimental procedures used in this protocol were approved by the Yale University Institutional Animal Care and Use Committee (IACUC, 2017&#8211;10557) and were in accordance with all federal policies and guidelines governing the use of vertebrate animals. All animal experiments were carried out on Ambystoma mexicanum (axolotls) in facilities at Yale University.
1. Diploid Embryo Generation
<ol>
	<li>Obtain GFP+ diploid embryos to serve as graft hosts through natural mating using one or two gfp parents24.</li>
	<li>Collect freshly laid diploid eggs and place them in a metal sieve.</li>
	<li>Rinse the eggs thoroughly with 40% Holtfreter&#39;s solution (20 mM NaCl, 0.2 mM KCl, 0.8 mM NaHCO3, 0.2 mM CaCl2, 4 mM MgSO4, pH to 7.4).</li>
	<li>Place the eggs in fresh 40% Holtfreter&#39;s solution and store at 12 &#176;C. 
	NOTE: Diploid embryos should always be obtained before moving on to haploid embryo generation.</li>
</ol>
2. Haploid Embryo Generation
<ol>
	<li>Female gamete donor preparation
	<ol>
		<li>48 h before conducting in vitro activation, anesthetize a sexually mature white or white/RFP female axolotl by immersion in 1 g/L HEPES-buffered MS-222 in 40% Holtfreter&#39;s solution25.</li>
		<li>Ensure that the animal is fully anesthetized after approximately 30 min of immersion by firmly pinching its tail between the thumb and forefinger. Fully anesthetized animals will not physically respond to any pinch.</li>
		<li>Prepare a solution of human chorionic gonadotropin (HCG) containing 10,000 U/mL in sterile saline.</li>
		<li>Using a 30 G insulin syringe, inject 0.15 CC of HCG (1,500 units) into the musculature dorsal to the hind limb of the anesthetized female at 45&#176; angle to the midline to avoid contact with the spinal cord.</li>
		<li>Return the female to fresh 40% Holtfreter&#39;s solution and place in an 8&#8722;12 &#176;C refrigerator.</li>
		<li>After 48 h, return the female to room temperature. Place a few stones or plastic plants in her container as surfaces for egg laying. 
		NOTE: Females injected with HCG will often deposit empty jelly cases for several hours before laying eggs.</li>
		<li>Remove the stones or plastic plants after the female has begun consistently laying eggs.</li>
		<li>Allow the female to sit in the empty tank for 30 min to 1 h. 
		NOTE: Withholding materials for the animal to lay eggs on will allow tighter control of oocyte collection.</li>
	</ol>
	</li>
	<li>Male gamete collection
	<ol>
		<li>While the female&#39;s egg laying is stalled, anesthetize a sexually mature GFP+ male axolotl to serve as a sperm donor, as in step 2.1.1.</li>
		<li>Place the fully anesthetized male on his back on damp paper towels under a dissecting microscope.</li>
		<li>If the experimenter is righthanded, position the tip of a P1000 pipette at the base of the cloaca with the right hand.</li>
		<li>Place the left forefinger and thumb of the left hand 2&#8722;3 cm rostral to the pelvis. Gently squeeze the animal while moving the fingers towards the hind legs to flush out spermic urine samples.</li>
		<li>Collect each that is flushed out of the cloaca into an individual microcentrifuge tube. Repeat this process to collect 6 to 10 samples.</li>
		<li>Pipette 5.0 &#181;L from each sample onto a petri dish to inspect the quality of the sperm using an inverted microscope. 
		NOTE: Concentrated sperm samples are a milky white color, typically range from 5 to 20 &#181;L, and are usually retrieved after several, higher volume spermic urine samples are extracted. Sperm will collect at the bottom of the tubes in higher volume samples when left undisturbed. Concentrated samples of healthy sperm are highly motile and lose activity as their concentration decreases. Healthy males can produce up to 50 &#181;L of concentrated sperm.</li>
	</ol>
	</li>
	<li>Female gamete collection
	<ol>
		<li>After obtaining and confirming a healthy sperm sample, anesthetize the HCG-injected female axolotl as in step 2.1.1.</li>
		<li>Place the fully anesthetized female on damp paper towels on her back.</li>
		<li>Extract unfertilized eggs from the female using a hand motion similar to that in step 2.2.4.</li>
		<li>Collect the eggs using wet forceps and transfer them to a 10 cm petri dish. Treat eggs with irradiated sperm within 15 min of collection.</li>
	</ol>
	</li>
	<li>Male gamete preparation and in vitro activation
	<ol>
		<li>Use a P10 or P20 pipette to pipette the sperm up and down, breaking apart the clumps to form a homogenous suspension.</li>
		<li>Approximate the percentage of motile sperm by placing a 0.5 &#181;L drop of undiluted sperm suspension on a petri dish lid or glass slide and examining with an inverted microscope.</li>
		<li>Add 9.5 &#181;L of 0.1x Marc&#39;s modified Ringer&#39;s solution (MMR; Table of Materials) to this sample to make a 20x sperm dilution. Gently pipette up and down to mix.</li>
		<li>With an inverted microscope, count the sperm in three 1.0 &#181;L drops of the 20x diluted aliquot on a petri dish cover or hemocytometer to estimate the sperm concentration of the undiluted suspension.</li>
		<li>Prepare the sperm for irradiation by diluting an aliquot of the original sperm sample to about 80,000 motile cells/mL in sterile 0.1x MMR.</li>
		<li>Count the number of eggs obtained within the past 15 min. Add 0.5 &#181;L of the freshly diluted sperm from step 2.4.5 per egg to a petri dish. Use the pipette tip to spread this suspension into a 1 mm thick layer.</li>
		<li>Using a plastic lift, place the sample 4 cm from the bulbs of a 254 nm crosslinker. Genetically inactivate the sperm by irradiating the sample with 800,000 uJ/mm2.</li>
		<li>Using a P10 pipette, pipette the suspension onto the unfertilized eggs, coating each egg with 0.25&#8722;0.5 &#181;L of irradiated sperm. Allow the eggs to sit at room temperature for 30 min.</li>
		<li>After 30 min, flood the eggs with 0.1x MMR. Immediately place the eggs in an 8&#8722;10 &#176;C incubator for injections the next day or at 18 &#176;C for injections within 7 hours post activation (hpa).</li>
		<li>Dejelly haploids using sharp forceps 30 min after hydration. Immediately place the eggs in an 8&#8722;10 &#176;C incubator for injections the next day or at 18 &#176;C for injections the same day.</li>
	</ol>
	</li>
</ol>
3. Haploid Mutagenesis and Maintenance
<ol>
	<li>CRISPR/Cas9 microinjections
	<ol>
		<li>Design sgRNAs using CRISPRscan and synthesize26,27.</li>
		<li>For multiplex mutagenesis, prepare a stock of 5 sgRNAs (10 ng/&#181;L for each sgRNA) and Cas9 protein (1 &#181;g/&#181;L). Prepare a 100-fold dilution of this stock (0.1 ng/&#181;L per sgRNA, 10 ng/&#181;L Cas9) for injection. For single gene, high mutation frequency mutagenesis, follow the protocol outlined previously28.</li>
		<li>Inject haploid embryos at the single cell stage 7 hpa if stored at 18 &#176;C or inject embryos the next day at the 2&#8722;8 cell stage if they are stored at 8&#8722;10 &#176;C.</li>
		<li>Transfer the embryos to 1.0x MMR with 20% polysucrose 400.</li>
		<li>If single cell, inject each embryo with a 5 nL drop of the injection solution (approximately &#188; radius of the egg) containing a total mass of 0.5 pg/sgRNA and 50 pg Cas9 protein. If multicellular, distribute this mass by injecting smaller volumes into multiple cells.</li>
		<li>Allow the embryos to heal in the polysucrose 400 for a minimum of 4 h and a maximum of 18 h at 18 &#176;C.</li>
	</ol>
	</li>
	<li>Haploid embryo housing
	<ol>
		<li>Transfer the embryos to 0.1x MMR with antibiotic-antimycotic.</li>
		<li>House each embryo in an individual well of a 24-well plate, as some will die or develop abnormally. Maintain at 16&#8722;18 &#176;C. Lower temperatures can be used to prolong development, if necessary.</li>
		<li>Replace the media with fresh 0.1x MMR with antibiotic-antimycotic every other day.</li>
	</ol>
	</li>
</ol>
4. Diploid Host Embryo Preparation
<ol>
	<li>Maintain embryos in the jelly coating at 12 to 16 &#176;C until they reach stage 22 to 26.</li>
	<li>Collect the embryos that are ready for grafting, place them within a sieve (4 mm mesh size), and gently rinse them with 40% Holtfreter&#39;s solution.</li>
	<li>Transfer the embryos into filter-sterilized 0.1x MMR containing 1.5% bleach for up to 2 min. Completely submerge the embryos in the bleach solution and swirl them gently to ensure that the jelly coating makes full contact with the bleach solution to kill the microbes present on the embryos.</li>
	<li>After 2 min, dilute the bleach solution containing the embryos with an equal volume of filter-sterilized 0.1x MMR.</li>
	<li>Gently pour the embryos into a bleach-sanitized sieve (4 mm mesh size) and rinse the embryos five times with filter-sterilized 0.1x MMR.</li>
	<li>Place the embryos into sterile 10 cm petri dishes with 0.1x MMR with antibiotics for dejellying (penicillin 100 units/mL, streptomycin 100 &#181;g/&#181;L, 0.25 &#181;g/mL, gentamicin 25 &#181;g/mL).</li>
	<li>Under a fluorescent stereomicroscope, remove the jelly coats and vitelline membranes from GFP+ embryos using sharp forceps (tip dimensions 0.05 x 0.01 mm2).</li>
	<li>Transfer the GFP+ embryos to a new petri dish. Minimize the amount of liquid transferred from the petri dish where they were dejellied.</li>
	<li>Rinse the GFP+ host embryos with sterile 0.1x MMR with antibiotics four to six times in order to remove contaminants.</li>
	<li>Place the clean embryos at 4 &#176;C overnight before grafting. 
	NOTE: Cooling the embryos makes them more rigid and clean separation of the mesoderm from the endoderm feasible.</li>
</ol>
5. Haploid-diploid Chimera Generation
<ol>
	<li>Surgical dish and media preparation
	<ol>
		<li>Prepare sterile surgical operating dishes by pouring autoclaved 2% agarose in 0.1x MMR into sterile 35 mm easy-grip petri dishes. Fill the petri dishes halfway with agarose.</li>
		<li>After the agarose cools, use a sterile scalpel to cut a 25 mm long, slanted trough in the agarose to hold the embryos in place.</li>
		<li>Fill the dish with sterile surgical media (0.1x MMR with anti-mycoplasma 2.5 &#181;g/mL, amphotericin B 0.25 &#181;g/mL, and ciprofloxacin 10.0 &#181;g/mL) and refrigerate at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Embryo grafting procedure
	<ol>
		<li>Place one healthy haploid donor with one or two stage-matched GFP+ diploid host embryos inside the trough of the prechilled operating dish containing surgical media (Figure 3). 
		NOTE: Perform the procedure on a cooling stage (10 &#176;C or lower) if possible.</li>
		<li>Use two ultra-fine, autoclaved forceps (straight tip, tip dimensions 0.05 x 0.02 mm2) to remove the ectoderm and mesoderm layers from the host with the limb bud near the center (Figure 4). 
		NOTE: The rectangular tissue graft region encompasses the limb bud and extend from the ninth somite to the posterior half of the gill bulge, about 2 mm, along the anterior posterior axis. Along the dorsoventral axis, the grafted region spans approximately 1.5 mm, including the somites to just beyond the ventral edge of the gill bulge. The grafted region includes all lateral plate mesoderm and the lateral halves of the somites, without disturbing underlying endoderm. See Figure 4 and the accompanying video for details.</li>
		<li>Set aside the host tissue and remove an equivalently sized tissue sheet from the haploid donor using the same methods.</li>
		<li>Place the haploid donor tissue sheet onto the corresponding region of the donor embryo.</li>
		<li>Secure the tissue by covering it with an autoclaved, rectangular glass shard from a crushed microscope cover glass and gently pressing it into the host embryo body.</li>
		<li>Flip the haploid donor embryo onto its other side to harvest the limb bud for the second host embryo. Repeat steps 5.2.2 through 5.2.5.</li>
		<li>Carefully remove the remaining excess host tissues and the donor embryo from the dish.</li>
		<li>Leave the grafts with the glass shard anchors in place for 60&#8722;75 min, checking every 20 min to ensure that the glass has not slipped off.</li>
		<li>After the tissue grafts fully adhere, use the forceps to slowly peel off the glass shard anchors.</li>
	</ol>
	</li>
	<li>Chimera maintenance
	<ol>
		<li>Transfer the engrafted embryos to fresh surgical media and maintain them at 8&#8722;12 &#176;C overnight to heal. Individually house engrafted embryos in 12 or 24-well plates.</li>
		<li>After 36&#8722;48 h, transfer the engrafted embryos to sterile 0.1x MMR antibiotic-antimycotic. The strong antibiotics in the surgical media cause toxicity in engrafted embryos after 3 days.</li>
		<li>Replace the media with fresh 0.1x MMR and antibiotics every 2&#8722;3 days.</li>
		<li>Maintain the engrafted embryos at 18 &#176;C until they are able to feed28.</li>
		<li>After 1 to 2 months of development and care, haploid limbs can be scored for purity of the graft using a fluorescent dissection microscope. 
		NOTE: The presence of non-neural or non-blood host-derived GFP-tissue in limbs is an indicator of impure grafting and is often associated with abnormal limb development. These animals should be excluded from further analysis.</li>
	</ol>
	</li>
</ol>

<ol><li>Kragl, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cells+keep+a+memory+of+their+tissue+origin+during+axolotl+limb+regeneration%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cells keep a memory of their tissue origin during axolotl limb regeneration.</a> Nature. 460 (7251), 60-65 (2009).</li>
<li>Maden, M., Goodwin, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experiments+on+developing+limb+buds+of+the+axolotl+Ambystoma+mexicanum%5BTitle%5D)+AND+%22Journal+of+Embryology+and+Experimental+Morphology%22%5BJournal%5D)">Experiments on developing limb buds of the axolotl Ambystoma mexicanum.</a> Journal of Embryology and Experimental Morphology. 57, 177-187 (1980).</li>
<li>McCusker, C. D., Diaz-Castillo, C., Sosnik, J., Phan, A. Q., Gardiner, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cartilage+and+bone+cells+do+not+participate+in+skeletal+regeneration+in+Ambystoma+mexicanum+limbs%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Cartilage and bone cells do not participate in skeletal regeneration in Ambystoma mexicanum limbs.</a> Developmental Biology. 416 (1), 26-33 (2016).</li>
<li>Brun, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+analysis+of+the+eyeless+mutant+in+the+mexican+axolotl+%28Ambystoma+mexicanum%29%5BTitle%5D)+AND+%22Integrative+and+Comparative+Biology%22%5BJournal%5D)">Experimental analysis of the eyeless mutant in the mexican axolotl (Ambystoma mexicanum).</a> Integrative and Comparative Biology. 18 (2), 273-279 (1978).</li>
<li>Lopez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+hematopoiesis+in+a+fully+regenerative+vertebrate%3A+the+axolotl%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl.</a> Blood. 124 (8), 1232-1242 (2014).</li>
<li>de Both, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+Axolotl+Heads%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Transplantation of Axolotl Heads.</a> Science. 162 (3852), 460-461 (1968).</li>
<li>Harrison, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Some+Unexpected+Results+of+the+Heteroplastic+Transplantation+of+Limbs%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Some Unexpected Results of the Heteroplastic Transplantation of Limbs.</a> Proceedings of the National Academy of Sciences of the United States of America. 10 (2), 69-74 (2006).</li>
<li>Fields, E., French, V., Bryant, P. J., Bryant, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((V+Pattern+regulation+in+epimorphic+fields%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">V Pattern regulation in epimorphic fields.</a> Science. 193 (4257), 969-981 (2013).</li>
<li>Gerber, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+analysis+uncovers+convergence+of+cell+identities+during+axolotl+limb+regeneration%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration.</a> Science. 362 (6413), (2018).</li>
<li>Knapp, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+transcriptional+profiling+of+the+axolotl+limb+identifies+a+tripartite+regeneration-specific+gene+program%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Comparative transcriptional profiling of the axolotl limb identifies a tripartite regeneration-specific gene program.</a> PloS One. 8 (5), e61352(2013).</li>
<li>Campbell, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+profile+of+the+regeneration+epithelium+during+axolotl+limb+regeneration%5BTitle%5D)+AND+%22Developmental+Dynamics%3A+an+official+publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">Gene expression profile of the regeneration epithelium during axolotl limb regeneration.</a> Developmental Dynamics: an official publication of the American Association of Anatomists. 240 (7), 1826-1840 (2011).</li>
<li>Bryant, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Tissue-Mapped+Axolotl+De+Novo+Transcriptome+Enables+Identification+of+Limb+Regeneration+Factors%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors.</a> Cell Reports. 18 (3), 762-776 (2017).</li>
<li>Gardiner, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+during+the+first+28+days+of+axolotl+limb+regeneration+I%3A+Experimental+design+and+global+analysis+of+gene+expression%5BTitle%5D)+AND+%22Regeneration%22%5BJournal%5D)">Gene expression during the first 28 days of axolotl limb regeneration I: Experimental design and global analysis of gene expression.</a> Regeneration. 2 (3), 120-136 (2015).</li>
<li>Flowers, G. P., Timberlake, A. T., McLean, K. C., Monaghan, J. R., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+targeted+mutagenesis+in+axolotl+using+Cas9+RNA-guided+nuclease%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease.</a> Development. 141 (10), Cambridge, England. 2165-2171 (2014).</li>
<li>Smith, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Chromosome-Scale+Assembly+of+the+Enormous+%2832+Gb%29+Axolotl+Genome%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">A Chromosome-Scale Assembly of the Enormous (32 Gb) Axolotl Genome.</a> bioRxiv. , 373548(2018).</li>
<li>Nowoshilow, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+axolotl+genome+and+the+evolution+of+key+tissue+formation+regulators%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The axolotl genome and the evolution of key tissue formation regulators.</a> Nature. 559 (7712), 50-55 (2018).</li>
<li>Fankhauser, B. Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Effects+of+Changes+in+Chromosome+Number+on+Amphibian+Development%5BTitle%5D)+AND+%22The+Quarterly+Review+of+Biology%22%5BJournal%5D)">The Effects of Changes in Chromosome Number on Amphibian Development.</a> The Quarterly Review of Biology. 20 (1), 20-78 (1945).</li>
<li>Malacinski, G. M., Brothers, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutant+Genes+in+the+Mexican+Axolotl%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Mutant Genes in the Mexican Axolotl.</a> Science. 184 (4142), 1142-1147 (1974).</li>
<li>Armstrong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gynogenesis+in+the+mexican+axolotl%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Gynogenesis in the mexican axolotl.</a> Genetics. 83 (4), 783-792 (1976).</li>
<li>Flowers, G. P., Sanor, L. D., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lineage+tracing+of+genome-edited+alleles+reveals+high+fidelity+axolotl+limb+regeneration%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Lineage tracing of genome-edited alleles reveals high fidelity axolotl limb regeneration.</a> eLife. 6, 1-15 (2017).</li>
<li>Shalem, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+-+scale+CRISPR+-+Cas9+knockout+screening+in+human+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genome - scale CRISPR - Cas9 knockout screening in human cells.</a> Science. 343 (6166), 84-87 (2014).</li>
<li>Wang, T., Wei, J. J., Sabatini, D. M., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+Screens+in+Human+Cells+Using+the+CRISPR-Cas9+System%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genetic Screens in Human Cells Using the CRISPR-Cas9 System.</a> Science. 343 (6166), 80-84 (2014).</li>
<li>Yin, Z., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+Meets+Single%3A+The+Application+of%5BTitle%5D)+AND+%22CRISPR%2FCas9+in+Haploid+Embryonic+Stem+Cells.+Stem+Cells+International%22%5BJournal%5D)">Simple Meets Single: The Application of.</a> CRISPR/Cas9 in Haploid Embryonic Stem Cells. Stem Cells International. 2017, 1-6 (2017).</li>
<li>Khattak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+axolotl+%28Ambystoma+mexicanum%29+husbandry%2C+breeding%2C+metamorphosis%2C+transgenesis+and+tamoxifen-mediated+recombination%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination.</a> Nature Protocols. 9 (3), 529-540 (2014).</li>
<li>Vachon, P., Zullian, C., Dodelet-Devillers, A., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+anesthetic+effects+of+MS222+in+the+adult+Mexican+axolotl+%28Ambystoma+mexicanum%29%5BTitle%5D)+AND+%22Veterinary+Medicine%3A+Research+and+Reports%22%5BJournal%5D)">Evaluation of the anesthetic effects of MS222 in the adult Mexican axolotl (Ambystoma mexicanum).</a> Veterinary Medicine: Research and Reports. 7, 1-7 (2016).</li>
<li>Montague, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+Mutagenesis+by+Cas9+Protein-Mediated+Oligonucleotide+Insertion+and+Large-Scale+Assessment+of+Single-Guide+RNAs%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs.</a> PLoS One. 9 (5), (2014).</li>
<li>Moreno-Mateos, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPRscan%3A+designing+highly+efficient+sgRNAs+for+CRISPR-Cas9+targeting+in+vivo%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.</a> Nature Methods. 12 (10), 982-988 (2015).</li>
<li>Kumar, A., Simon, A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKumar%2C+A+%22Salamanders+in+Regeneration+Research%3A+Methods+and+Protocols%22">Salamanders in Regeneration Research: Methods and Protocols</a>. , Humana Press. New York, NY. (2015).</li>
<li>Hronowski, L., Gillespie, L. L., Armstrong, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+Survival+of+Haploids+of+the+Mexican+Axolotl%2C+Ambystoma+mexicanum%5BTitle%5D)+AND+%22Journal+of+Experimental+Zoology%22%5BJournal%5D)">Development and Survival of Haploids of the Mexican Axolotl, Ambystoma mexicanum.</a> Journal of Experimental Zoology. 209, 41-47 (1979).</li>
<li>Schreckenberg, G. M., Jacobson, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+stages+of+development+of+the+axolotl%2C+Ambystoma+mexicanum%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Normal stages of development of the axolotl, Ambystoma mexicanum.</a> Developmental Biology. 42 (2), 391-399 (1975).</li>
<li>Hertwig, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beitrage+Zum+Determinations-+Und+Regenerationsproblem+Mittels+Der+Transplantation+Haploidkerniger+Zellen%5BTitle%5D)+AND+%22Archiv+f.+Entwicklungsmechanik%22%5BJournal%5D)">Beitrage Zum Determinations- Und Regenerationsproblem Mittels Der Transplantation Haploidkerniger Zellen.</a> Archiv f. Entwicklungsmechanik. 111, 292-316 (1927).</li>
<li>Fei, J. -F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+knockin+in+axolotl+and+its+use+to+test+the+role+of+satellite+cells+in+limb+regeneration%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (47), 12501-12506 (2017).</li>
</ol>

The authors have nothing to disclose.

There are a few critical steps in our protocol for generating haploid-diploid chimeras that the operating technician should consider for consistent grafting results.
The most likely reason for haploid generation to fail is due to poor in vitro activation conditions. The proper quantities of motile sperm must be used to activate eggs. To prolong motility, sperm samples should always be maintained at 4 &#176;C. Before applying any sperm sample to eggs, check the viability of the sperm using an i...

Historically, embryonic tissue grafting in amphibians has been an important technique for exploring fundamental mechanisms of developmental biology and regeneration. The axolotl, a species of salamander, possesses an impressive ability to regenerate tissues and complex structures such as limbs and organs after injury or amputation. Similarly impressively, they can receive, without rejection, tissue grafts from other individuals at embryonic, juvenile, and adult stages1,2,3. Regions of embryos that produce whole structures such as limbs, tails, eyes, and heads, and more specific tissues, such as neuroectoderm and somites, can be grafted between embryos to produce chimeric animals1,2,4,5,6. For nearly a century, studies of such chimeric animals have provided crucial insights into regeneration, tissue differentiation, size control, and patterning1,7,8.
In the last the decade, numerous transcriptional studies of regenerating tissues have produced insights into the genetic programs underlying salamander regeneration9,10,11,12,13. These studies have added to an expanding list of candidate genes that, to date, are largely uncharacterized in the context of regeneration. Targeted mutagenesis techniques, such as CRISPR/Cas, now permit the investigation of such genes, and such genetic approaches are greatly facilitated by the recent sequencing and assembly of the large axolotl genome14,15,16.
We sought to develop techniques that coupled classic developmental biology with new genetic technology for the purpose of dissecting the mechanisms of regeneration. Methods for generating haploid embryos of axolotls and other salamanders have been established for decades17. While these techniques have long been noted to be advantages of salamanders as genetic model organisms18, few subsequent genetic studies have incorporated haploid animals. We use in vitro activation in the axolotl to produce haploid embryos that serve as tissue donors for grafting19. Using embryos carrying fluorescent genetic markers, we have devised reliable methods for generating limbs derived almost entirely from donor tissues (Figure 1A). By combining these two techniques, we have bypassed the late embryonic lethality associated with haploidy, allowing for the production of fully developed, grafted haploid limbs (Figure 1B, Figure 1B&#39;, and Figure 2).
By conducting CRISPR/Cas-mediated mutagenesis in haploid embryos prior to grafting to create chimeric axolotls with mutant haploid limbs, we may investigate gene function specifically within the context of limb development and regeneration. This allows the rescue of limbs from potentially embryonic-lethal mutant phenotypes. While CRISPR/Cas microinjection can generate animals that are highly mutant, such animals are typically highly mosaic, with some degree of retention of wildtype alleles and a variety of distinct mutations at targeted sites14,20. CRISPR-based mutagenesis in haploid cells increases the penetrance of single allele loss-of-function mutations, as they cannot be masked by retained wildtype alleles. For this reason, CRISPR-based screening in haploid cell lines is increasingly used to investigate the genetic basis of many cellular processes21,22,23. By combining CRISPR-based lineage tracing with our haploid limb bud grafting protocols, the approach described here can serve as a platform for haploid genetic screens in living animals20.

<table><tbody><tr><td>#55 Dumont Forceps</td><td>Fine Science Tools</td><td>11295-1</td><td>Only use Dumostar material (can be autoclaved)</td></tr><tr><td>Amphotericin B</td><td>Sigma Aldrich</td><td>A2942-20ML</td><td>20 mL</td></tr><tr><td>Antibiotic-Antimycotic 100x</td><td>Thermo Fisher</td><td>15240062</td><td></td></tr><tr><td>Ciprofloxacin</td><td>Sigma Aldrich</td><td>17850-5G-F</td><td></td></tr><tr><td>Ficoll 400 (polysucrose 400)</td><td>bioworld</td><td>40600032-3</td><td>Ficoll 400</td></tr><tr><td>Gentamicin</td><td>Sigma Aldrich</td><td>G1914-250MG</td><td></td></tr><tr><td>Heating/Cooling Incubator</td><td>RevSci</td><td>RS-IF-233</td><td></td></tr><tr><td>Human Chorionic Gonadotropin</td><td>Merk</td><td></td><td>Chorulon</td></tr><tr><td>Megascript T7 Transcription Kit</td><td>Thermo Fisher</td><td>AM1334</td><td>40 reactions</td></tr><tr><td>Miroscope Cooling Stage</td><td>Brook Industries</td><td>Custom</td><td>Custom</td></tr><tr><td>NLS Cas9 Protein</td><td>PNAbio</td><td>CP01-200</td><td>4 vials of 50 &amp;micro;g protein each</td></tr><tr><td>Plasmocin</td><td>Invivogen</td><td>ant-mpt-1</td><td>Treatment level</td></tr><tr><td>&lt;strong&gt;Recipes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.0x Marc&#039;s modified Ringer&#039;s solution (MMR)</td><td></td><td></td><td>0.1 M NaCl, 2 mM KCl, 1 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 2 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.1 mM EDTA, 5 mM HEPES (pH 7.8), ph 7.4</td></tr><tr><td>40% Holtfreter&#039;s solution</td><td></td><td></td><td>20 mM NaCl, 0.2 mM KCl, 0.8 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 0.2 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 4 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, pH to 7.4</td></tr></tbody></table>

generation of chimeric axolotls with mutant haploid limbs through embryonic grafting

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.

Developing haploid embryos can be distinguished from diploid embryos by their 'haploid syndrome' phenotype29. At graft-stage, haploid embryos exhibit reduced curvature along the anterior-posterior axis and incomplete enclosure of the yolk plug (Figure 3A). A fluorescent microscope can be used to ensure that haploid embryos are free of paternally derived GFP expression (Figure 3B</s...

This goal of this protocol is to produce chimeric axolotls with haploid forelimbs derived from Cas9-mutagenized donor tissue using embryonic tissue grafting techniques.

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as ...

generation-chimeric-axolotls-with-mutant-haploid-limbs-through

Research

JoVE Journal

Genetics

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

Generating Chimeric Zebrafish Embryos by Transplantation

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras.  This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Recombinant limbs are a powerful experimental model that allows for studying the process of cell differentiation and the generation of patterns under the influence of embryonic signals. This protocol presents a detailed method for generating recombinant limbs with chicken limb-mesodermal cells, adaptable to other cell types obtained from different organisms.

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic amphibians.

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

In this protocol, we show how to prepare axolotl tissue for atomic force microscopy (AFM) and perform indentation measurements in intact and regenerating limb cartilage.

Atomic Force Microscopy Measurements of Cartilage in Intact and Regenerating Axolotl Limbs

Learn embryonic grafting techniques to generate chimeric axolotls with mutant haploid limbs for genetic screening. This method enables CRISPR-Cas9 mutagenesis studies and rescues embryonic lethal phenotypes.

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

Explore More Videos