THE JOURNAL OF EXPERIMENTAL ZOOLOGY 228~355-362(1983) Nuclear Transplantation in Mouse Embryos JAMES McGRATH AND DAVOR SOLTER The Wistar Institute of Anatomy and BLology, Philadelphia, Pennsylvania 19104 ABSTRACT The ability of foreign nuclei to support development in nuclear transplantation manipulations has proven an effective means to assess the consequences of nuclear differentiation. In addition, nuclear transplantation might serve to define the persistence and role of maternally inherited cyto- plasmic constitutents during embryogenesis. We have extended the use of a technique that enables the efficient transfer of one-cell-stage pronuclei into the cytoplasm of enucleated mouse embryos, and have successfully transferred two-, four-, eight-cell-stage and inner cell mass (ICM) cell nuclei. We have also used this technique as a means to determining that the stage-specific embry- onic antigen, SSEAS, is a cytoplasmic contribution of the unfertilized ovum. The potential value of this technique in determining the developmental capac- ity of nuclei from various embryonic stages, and in determining nuclear1 cytoplasmic origins of early embryonic gene products, is discussed. Key words nuclear transfer, mouse embryo, surface antigens The transplantation of foreign nuclei into the amphibian embryo demonstrated the varied abilities of embryonic and adult nu- clei to support development (Danielli and DiBerardino, '79). More recently, the ability of transplanted early embryonic nuclei from inner cell mass cells of the mouse blastocyst to support development has been reported (Illmensee and Hoppe, '81; Hoppe and 111- mensee, '82). These experiments have pro- vided valuable information regarding the status of nuclear differentiation at distinct stages of embryogenesis. A frequent diffi- culty encountered in microsurgical manipu- lations of the mammalian embryo, however, is the injury of a significant proportion of embryos due to micropipette penetration. To avoid this difficulty, we have devised a nu- clear transplantation technique which does not require micropipette penetration of the embryo plasma membrane (McGrath and Solter, '83). This method allows the removal of the embryonic pronuclei within a mem- brane-bound vesicle. The subsequent intro- duction ofa donor nucleus into the enucleated embryo is achieved using a virus-mediated cell fusion technique. This procedure enables nuclear transplantation to be reproducibly performed, a t a high frequency of success, and with little or no effect on subsequent development. This report presents applica- tions of this procedure in defining nuclear/ cytoplasmic interactions during mammalian embryogenesis, specifically, in defining the nuclearlcytoplasmic origin of an early em- bryonic antigen. MATERIALS AND METHODS Embryo isolation and culture Embryos were obtained from spontaneous inter se matings of C57BL6lJ, C3H/HeJ and ICR (Swiss Albino) strain mice. One-cell- stage embryos were removed from mated fe- males by puncturing the ampullary regions of excised oviducts with watchmaker forceps on the day of vaginal plug detection (day 1). Cumulus cells were removed with N-2- hydroxyethyl piperazine-N'-2-ethanesulfonic acid (Hepes)-buffered Whitten's medium (Whitten, '71) containing 500 NF unitdm1 bovine hyaluronidase (Sigma). Embryos were washed by transfer through successive drops (four) of modified Whitten's medium (Abram- czuk et al., '77) and cultured in this medium under silicone oil (Dow Corning 200 Fluid) as previously described (McGrath and Hillman, '80). Incubations were performed at 37°C in an atmosphere of 5% O2,5%CO2,and 90% Nz. Two-cell-stage and four- to eight-cell-stage embryos were flushed from the oviducts of mated females on gestational days 2 and 3, respectively. Blastocyst-stage embryos were flushed from the uteri of mated females (day 4) and cultured overnight. Inner cell mass 0 1983 ALAN R. LISS. INC.