ADENOVIRUS-MEDIATED GENE-TRANSFER BY PERIVITELLINE MICROINJECTION OF MOUSE, RAT, AND COW EMBRYOS

To determine the fate of an episomally expressed transgene, mouse, rat , and cow zygotes were injected into the perivitelline space with appr oximately 100 pl of buffer containing the replication-defective human adenovirus, AdCMVLacZ/sub360. Viral concentrations ranged from 2.5 to 2.5 x 10(5) plaque-forming units (pfu)/100 pl. As viral titer increase d, fewer embryos were able to develop to blastocysts. In the mouse, th e percentage of blastocysts formed ranged from 82% in controls to 16% after injection at the highest titer. In the rat and cow, a similar de crease in blastocyst formation was noted (62% to 6% and 26% to 4%, res pectively). Reporter gene (galactosidase, LacZ) activity could be dete cted in mouse embryos after injection at a concentration of only 25 pf u/100 pi, whereas a tenfold higher titer was required in the other two species to observe the blue LacZ reaction product. When examined afte r 5 (mouse), 6 (rat), or 9 (COW) days of in vitro culture, the proport ion of LacZ-positive embryos ranged from 15% to 96%, 6% to 76%, and 18 % to 58% in mouse, rat, and cow embryos, respectively, depending upon viral concentration. However, a large percentage of positive embryos p roved to be expression mosaics, the degree of which was likewise depen dent on titer. While none of the embryos showed LacZ activity at 30 h after injection, 70% of mouse, 8% of rat, and 20% of cow embryos expre ssed the reporter gene at 42 h. Delaying the timing of injection revea led that the efficiency with which mouse and rat embryos could be infe cted decreased with increasing degree of differentiation. Only 35% and 18% of mouse embryos expressed the reporter gene after injection at t he morula or blastocyst stage, respectively. A similar drop in efficie ncy was noted in rat embryos when injections took place at the 8-cell, morula, or blastocyst stage, with 70%, 33%, and 9% of embryos, respec tively, subsequently showing LacZ activity Likewise, advanced developm ent resulted in a decrease in the efficiency of viral-mediated gene tr ansfer in cow embryos, with 100%, 78%, and 68% of embryos being positi ve after injection at the 8-cell, morula, or blastocyst stage, respect ively. These results demonstrate that a human adenovirus can be used t o express a reporter gene transiently in nonhuman embryos.