Gynogenetic Production of Embryos in Xenopus tropicalis Using a Cold Shock Procedure: Rapid Screening Method for Gene Editing Phenotypes.

Abstract

Gynogenesis is a form of parthenogenesis in which eggs require sperm for fertilization but develop to adulthood without the contribution of paternal genome information, which happens naturally in some species. In Xenopus, gynogenetic diploid animals can be made experimentally. In mutagenesis strategies that only generate one allele of a recessive mutation, as might occur during gene editing, gynogenesis can be used to quickly reveal a recessive phenotype in eggs carrying a recessive mutation, thereby skipping one generation normally required to screen by conventional genetics. Xenopus oocytes do not complete meiosis until shortly after fertilization, and the second polar body is retained in fertilized eggs. Using ultraviolet (UV)-irradiated sperm, fertilization can be triggered without a genetic paternal contribution. Upon applying cold shock at the proper time to such embryos, ejection of the second polar body can be suppressed and both maternal sister chromatids are retained, leading to the development of gynogenetic diploid embryos. Because the genome of the resultant animals consists of recombined sister chromatids because of crossover events during meiosis, it is not completely homozygous throughout the whole genome. Nevertheless, the genome is homozygous at some loci proximal to the centromere that are unlikely to undergo recombination during meiosis and homozygous at reduced frequency if mutations are farther from the centromere, but still generally at a scorable level. Therefore, this technique is useful for rapid screening phenotypes of recessive mutations in such regions. We describe here a step-by-step protocol to achieve cold shock-mediated gynogenesis in Xenopus tropicalis.