Name: generation of chimeric axolotls with mutant haploid limbs through embryonic grafting
Uploaded: 2020-01-29T13:00:00
Description: Watch this Scientific Journal Video about Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting at JoVE.com

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 o...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+analysis+of+the+eyeless+mutant+in+the+mexican+axolotl+(Ambystoma+mexicanum).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+hematopoiesis+in+a+fully+regenerative+vertebrate:+the+axolotl.">Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl.</a> Blood. 124 (8), 1232-1242 (2014).</li><li>de Both, N. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Changes+in+Chromosome+Number+on+Amphibian+Development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+Genes+in+the+Mexican+Axolotl.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gynogenesis+in+the+mexican+axolotl.">Gynogenesis in the mexican axolotl.</a> Genetics. 83 (4), 783-792 (1976).</li><li>Flowers, G. P., Sanor, L. D., Crews, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Screens+in+Human+Cells+Using+the+CRISPR-Cas9+System.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Meets+Single:+The+Application+of.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+axolotl+(Ambystoma+mexicanum)+husbandry,+breeding,+metamorphosis,+transgenesis+and+tamoxifen-mediated+recombination.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+anesthetic+effects+of+MS222+in+the+adult+Mexican+axolotl+(Ambystoma+mexicanum).">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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;g protein each</td>
		</tr>
		<tr>
			<td>Plasmocin</td>
			<td>Invivogen</td>
			<td>ant-mpt-1</td>
			<td>Treatment level</td>
		</tr>
		<tr>
			<td>Recipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.0x Marc&#39;s modified Ringer&#39;s solution (MMR)</td>
			<td></td>
			<td></td>
			<td>0.1 M NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES (pH 7.8), ph 7.4</td>
		</tr>
		<tr>
			<td>40% Holtfreter&#39;s solution</td>
			<td></td>
			<td></td>
			<td>20 mM NaCl, 0.2 mM KCl, 0.8 mM NaHCO3, 0.2 mM CaCl2, 4 mM MgSO4, pH to 7.4</td>
		</tr>
	</tbody>
</table>

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 &#39;haploid syndrome&#39; 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 3<str...

Watch this Scientific Journal Video about Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting at JoVE.com

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

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 ...

This protocol allows the production of mutagenized haploid limbs that can be used for genetic screening in the axolotl. In haploids, even low-level mutagenesis can produce loss of function phenotypes within cells. This grafting technique can also be used to rescue embryonic lethal phenotypes.This method can be used to study regeneration in any amphibian species that is amenable to in vitro fertilization and grafting. Before using haploid embryos as donors, we recommend practicing and confirming the success of grafts between GFP positive and negative diploid embryos. For female gamete donor preparation, anesthetize a sexually mature white or white RFP female axolotl to serve as the gamete donor.After confirming a lack of response to pain simulation, use a 30 gauge insulin syringe to inject 0.15 milliliters of human chorionic gonadotropin into the musculature dorsal to the hind limb. Insert the syringe at a 45 degree angle to the midline to avoid contact with the spinal cord. Place the injected animal in fresh 40%Holtfreter's solution and transfer the axolotl to an eight to 12 degree Celsius refrigerator for 48 hours.Then return the female to room temperature. While the female is laying eggs, place a fully anesthetized male in the supine position on damp paper towels under a dissecting microscope and position the tip of a P1000 pipette at the base of the cloaca with the dominant hand. Place the other forefinger and thumb two to three centimeters rostral to the pelvis and gently squeeze the animal while moving the fingers toward the hind legs to flush out the spermic urine samples collecting each sample into a new microcentrifuge tube.Then transfer five microliters from each sample into a Petri dish to inspect the quality of the sperm using an inverted microscope. After counting, dilute the sperm to a concentration of eight times 10 to the fourth cells per milliliter in 0.1X sterile MMR and transfer 0.5 microliters of sperm cells per egg to a new Petri dish. Then use the pipette tip to spread the suspension to a one millimeter thick layer and use a plastic lift to place the sample four centimeters from the bulbs of a 254 nanometer crosslinker for irradiation at eight times 10 to the fifth microjoules per millimeter squared.After collecting a healthy sperm sample, place the female in the supine position on a stack of damp paper towels and use a similar flushing motion to extract unfertilized eggs from the female axolotl. Then use wet forceps to transfer the eggs into a 10 centimeter Petri dish. Within 15 minutes of collection, use a P10 pipette to dispense the irradiated sperm onto the unfertilized eggs coating each egg with 0.25 to 0.5 microliters of inactivated sperm.After allowing the eggs to sit at room temperature for 30 minutes, flood the eggs with 0.1X MMR. Thirty minutes after the hydration, use sharp forceps to dejelly the eggs and place the eggs at 18 degrees Celsius for microinjection within seven hours. To generate a haploid-diploid chimera, place one healthy haploid donor with one or two stage matched GFP positive diploid host embryos inside the trough of a pre-chilled operating dish containing surgical medium on a 10 degree Celsius or lower cooling stage.Use two ultra fine autoclaved straight tip forceps to remove the entire limb bud and the surrounding ectoderm and mesoderm tissue layers and set aside the host tissue. Remove an equivalently sized tissue sheath from the haploid donor and place the haploid donor tissue sheath onto the corresponding region of the host embryo. Secure the tissue with an autoclaved rectangular glass shard from a crushed microscope coverglass and gently press the tissue into the host embryo body.Leave the anchor in place for 60 to 75 minutes checking every 20 minutes to confirm that the glass has not slipped before using the forceps to peel off the coverglass fragment. Transfer the engrafted embryos into fresh surgical medium allowing them to heal at eight to 12 degrees Celsius overnight before housing the engrafted embryos individually in 12 or 24-well plates. 38.7%of oocytes developed into normally developed haploid embryos.At the graft stage, haploid embryos exhibit a reduced curvature along the anterior-posterior axis and an incomplete enclosure of the yolk plug. In addition, a fluorescent microscope can be used to confirm the haploid embryos are free of paternally-derived GFP expression. Failed and impure grafts produce a variety of phenotypes.When the grafts are clean and normally developed, GFP expression should be limited primarily to the brachial plexus, the neural network derived from the host spinal cord. Punctate GFP expression is also present in single cells that appear to be sensory neurons in blood-derived host cells that migrate into the developing limb. When RFP positive donors are used, haploid graft limbs exhibit a universal expression of RFP.Throughout development, non-mutagenized haploid limbs are significantly shorter than the opposing diploid forelimbs in chimeric animals. Non-mutagenized haploid limbs fully regenerate, although they demonstrate a slight delay in reaching the digital outgrowth stage compared to diploids. Remember to use sterile technique and completely remove and replace the full mesoderm and ectoderm tissue layers from the embryonic host in order to produce a cleanly grafted limb.Mutant limbs can be amputated and scored for regeneration phenotypes. Mutant alleles can be quantified with next generation sequencing. Combined with CRISPR-Cas9 mutagenesis, our grafting technique has allowed us to conduct a negative selection screen of targeted genes in regenerating haploid limbs.

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

Research

JoVE Journal

Genetics

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication ...

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.