In addition to describing a procedure for making transgenic frog embryos, we are also including a new protocol for using sperm nuclear transplantation to fertilize oocytes that have been matured in vitro. A number of investigators have successfully depleted maternal RNAs from Xenopus embryos by injecting oocytes with antisense deoxyoligonucleotides for a message of interest, and then maturing and fertilizing these injected oocytes to produce embryos (12-15). However, the approaches currently available for obtaining embryonic development from oocytes matured in vitro are quite labor-intensive and technically demanding (16). The difficulty arises in large part because the surface of matured oocytes must be altered by proteolytic enzymes, and covered with a jelly coat in the oviduct of the female frog in order to become competent for fertilization. When oocytes are matured in vitro, these processes must either be simulated, or the matured oocytes must be reimplanted into the body cavity of a female frog so that these processes occur as it travels back outside through the oviduct of the host female. However, we have found that direct transplantation of sperm nuclei into matured oocytes bypasses these requirements and has allowed us to very simply produce normal embryos from oocytes handled in vitro. A similar approach has also been developed to overcome male infertility in humans (17-20).
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