Culture and Host Transfer of Xenopus Oocytes for Maternal mRNA Depletion and Genome Editing Experiments.

Abstract

The early development of Xenopus critically depends on maternal components stored in the egg. Because important events such as axis formation are triggered immediately after fertilization, it is often desirable to perturb gene function before this occurs. Oocytes can be experimentally manipulated in vitro, prior to maturation, and subsequently fertilized or otherwise activated to develop, and then observed for any embryological defects. Available methods for fertilizing cultured oocytes include in vitro fertilization following oocyte vitelline envelope removal, nuclear transplantation, intracytoplasmic sperm injection, and transferring oocytes to the body cavity of ovulating host females (host transfer). This chapter outlines this host transfer method, which has been used to elucidate basic mechanisms of axis formation, germ-layer induction, and primordial germ cell specification. Methods for obtaining, culturing, transferring, and fertilizing Xenopus oocytes are described. This method has typically been used to alter maternal gene function by antisense oligonucleotide-mediated mRNA knockdown, but is also useful for mRNA or protein overexpression, including the expression of genome-editing reagents prior to fertilization.