CLONING Volume 2, Number 1, 2000 Mary Ann Liebert, Inc. Blastomeres from Somatic Cell Nuclear Transfer Embryos Are Not Allocated Randomly in Chimeric Blastocysts KEVIN D. WELLS and ANNE M. POWELL ABSTRACT A marker has been developed to allow detection of blastomeres that originate from embryos produced by nuclear transfer (NT) of genetically engineered fetal fibroblasts. A plasmid (phEFnGFP) was constructed with a G418 resistance cassette for selection in fibroblasts and a nuclear localized green fluorescent protein (nGFP) expression cassette that expresses in every cell of day-6, -7, and -8 bovine embryos. This construct was utilized to follow the blastomere distribution in aggregation chimeras produced from fertilized embryos (in vitro produced, IVP) or parthenotes and NT embryos. Fluorescent and nonfluorescent NT embryos were ag- gregated early on day 4 and evaluated on day 8. Nuclei of blastomeres that carried the trans- gene were fluorescent under both UV epifluorescence (Hoechst 33342) and blue epifluores- cence (nGFP). There was no bias in the distribution of green fluorescent blastomeres in the inner cell mass (ICM) or trophectoderm in NT,. NT chimeras. However, there was a strong bias for NT blastomeres to populate the ICM when aggregated with IVP embryos or parthenotes. There was also a strong bias against NT blastomeres in the trophectoderm when aggregated to IVP embryos. However, the bias against NT blastomeres in the trophectoderm was significantly less (p , 0.05) when aggregated with parthenotes as compared to aggrega- tion with IVP embryos. In NT,. NT aggregates, no chimeric embryos were produced that had an ICM composed of blastomeres from a single origin. However, in NT,. Parthenote ag- gregates, 67% of the blastocysts had an ICM composed exclusively of NT origin. The re- maining blastocysts ranged from 0% to 83% of the ICM that expressed nGFP. Similarly, in NT,. IVP aggregates 50% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 19% to 71% of the ICM being of NT origin. We con- clude that production of divaricated chimeras from NT origin is feasible. Other applications of this technology are discussed. 9 INTRODUCTION S IMILAR TO MOUSE EMBRYONIC STEM (ES) CELLS for murine models, secondary somatic cell cul- tures now promise the ability to precisely ma- nipulate the genome of livestock. By utilizing the limited period available for in vitro culture be- tween cell collection and senescence, somatic cells can be genetically modified by standard tissue culture techniques and then selected for particu- lar phenotypes and/or genotypes. After assess- ment of individual integration events, cells from Gene Evaluation and Mapping Laboratory, ARS, USDA, Beltsville, Maryland.