Timing of DNA integration, transgenic mosaicism, and pronuclear microinjection

Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.