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.

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.