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

The overall goal of leapfrogging is to facilitate the creation of mutant lines for genes that are essential for embryogenesis. This method can help answer key questions in Developmental Genetics and other fields. The main advantage of this technique is that one can obtain mutants in essential genes in the F1 generation.

To begin this procedure, microinject to Xenopus embryos at the one cell stage with Cas9-sgRNA complexes as outlined in the text protocol. When the embryos reach 4.5 hours post-fertilization, at early blastula stage nine, remove them from the 25 degree Celsius incubator to allow them to continue development at room temperature. To begin the transplantation at five hours post-fertilization, use a pasteur pipette to transfer one uninjected embryo to a 60 millimeter agarose dish containing depressions, which will serve as the graft recipient.

Also transfer one PGC donor embryo to the dish. Use forceps to manually remove the vitelline envelopes from each embryo. And rotate both embryos so that their vegetal poles are in view and accessible for surgery.

Next, while stabilizing the recipient embryo with the hair loop, insert the sharp tip of an eyebrow hair knife into the vegetal pole, just below the surface. And with rapid, upwards slicing movements, make four shallow incisions in the shape of a square, inside the zone where future bottle cells will form that mark the blastopore. Once the four sides of the square are delineated, use the eyebrow hair knife to deepen each incision using similar cutting motions to a depth of approximately 1/3 to 1/2 the distance to the blastocoel floor.

Free the vegetal tissue explant from the recipient embryo by making one or more horizontal cuts in the deep vegetal region. Then, working quickly, repeat this procedure on the PGC donor embryo to create a similarly sized vegetal tissue fragment for transplantation. Once the tissue containing PGCs is removed from the donor embryo, use the eyebrow hair knife and hair loop to position the explant in the opening created in the recipient embryo.

Use the shaft of the eyebrow hair knife, held parallel to the surface of the graft, to gently press the graft into the opening in the vegetal surface of the recipient embryo. Use the hair loop to gently slide the carcass of the donor embryo across the agarose surface and into a well. Make sure that the open wound of the carcass is facing the bulk liquid.

Slide the recipient embryo into an adjacent well, and likewise, make sure that the grafted vegetal pole tissue is facing the bulk liquid. Repeat the transplantation of PGCs with additional embryos to create more transplant carcass pairs, until the embryos reach approximately early gastrula stage 10, transferring embryos to empty wells as the transplants are made. Once grafts have healed into place, typically 30 to 45 minutes after transplantation, use a pasteur pipette to very gently transfer embryos from the depressions to individual wells in an agarose-coated 24-well plate.

Rotate the embryos so that they are positioned with the vegetal pole up, facing the bulk solution. Place the donor carcasses and graft recipients in adjacent wells to assist in keeping track of embryo pairs and record this information. Then, transfer the 24-well plate containing embryos to a 25 degree Celsius incubator overnight.

The next day, move each graft recipient, now at the early tail bud stage, to an individual well of a fresh, agarose-coated, six-well plate. Raise the leapfrogged embryos and carry out analysis of donor DNA obtained from the carcasses according to the text protocol. Because leapfrogging produces animals that have efficient replacement of their germline with mutant alleles, mutant phenotypes can be obtained in the F1 generation.

As presented in this table, Blitz et al. reported that a majority of F0 animals, targeted at the tyrosinase locus, which is required for melanocyte and retinal pigementation, show 100%germline transmission of donor-derived mutant genomes, as measured by outcrossing with tyr minus albino animals. Having watched this video, you should have a good understanding of how to transplant Xenopus primordial germ cells.