[29] Microinjection into Xenopus oocytes

Much of the progress in understanding the cellular function of oncogenes derives from the ability to infect or transfect somatic cells in culture and to analyze the morphological and biochemical effects of expression of the introduced gene product. The amphibian oocyte, most notably that of Xenopus laevis, offers an alternative approach that overcomes many of the disadvantages of using somatic cells. Like somatic cells, Xenopus oocytes have been used very successfully for the transient expression of heterologous membrane receptor proteins and ion channels. The major features of Xenopus oocyte maturation and early embryonic development are presented in the chapter. Experiments designed to elucidate the function of the Mos and Ras proteins in Xenopus oocyte maturation offer examples of this technique. Mos was shown to be necessary for the initiation of meiotic maturation in Stage VI oocytes by injecting mos antisense oligonucleotides into progesterone-treated oocytes and preventing maturation. Moreover, injecting a soluble recombinant Mos protein into oocytes, in the absence of protein synthesis, demonstrated that Mos is sufficient for the induction of maturation-promoting factor (MPF) activation and germinal vesicle breakdown (GVBD). The Ras oncoprotein was also shown to induce progesterone-independent meiotic maturation in Xenopus oocytes. In addition, Ras, like Mos/CSF, arrests Xenopus embryonic cleavage after injection into one blastomere of a two-cell embryo. The chapter also describes the protocols that outline methods for the injection of synthetic mRNAs and purified proteins into oocytes, and include the embryonic cleavage arrest assay.