TRANSGENE DETECTION DURING EARLY MURINE EMBRYONIC-DEVELOPMENT AFTER PRONUCLEAR MICROINJECTION

The polymerase chain reaction (PCR) technique was used to detect a whe y acidic protein (WAP) gene and transgene presence in mouse ova cultur ed to various stages of development after pronuclear microinjection at the one-cell stage. The PCR technique detected an endogenous 442 bp W AP DNA sequence in 78% of one-cell, 88% of two-cell and 94% of four-ce ll ova, and in 95% of morulae and 97% of blastocysts. The heterologous WAP-human protein C transgene was detected in 88% of one-cell, 88% of two-cell and 44% of four-cell ova, and in 40% of morulae and 29% of b lastocysts. For comparison, the integration frequency for transgenic m ouse production using the same DNA construct was 22%. After five days of in vitro culture, embryos that were either developmentally arrested or fragmented were tested for the presence of the transgene. The inje cted construct was detected in 83% of arrested one-cell, 85% of arrest ed two-cell, and 85% of fragmented ova. In culture, only 28% of zygote s microinjected with DNA developed to the blastocyst stage compared to 74% of noninjected zygotes, while 63% of zygotes developed to the bla stocyst stage after injection of buffer alone. Pronuclear injection of the transgene at concentrations of 1.5, 15 and 50 mu g ml(-1) resulte d in 28, 11 and 9% development to blastocysts and 29, 86 and 88% trans gene detection, respectively. Transgene detection was 85, 96 and 97% i n degenerate embryos at the respective doses of DNA. These data show t hat pronuclear microinjection of the transgene is detrimental to subse quent embryonic development. Also, unintegrated copies of the transgen e probably exist at least until the blastocyst stage, and thereafter a re degraded to the extent that they can no longer be detected by PCR.