Developmental capacity of ovine zygotes following microinjection of h FVIII-cDNA

The mammary gland of transgenic livestock has recently been shown to be an appropriate site for the production of recombinant human pharmaceutical proteins in biologically active forms. The purpose of our experiments was to generate transgenic sheep in which the human blood clotting factor VIII gene (hFVIII) is expressed in the ovine mammary gland. A total of 920 zygotes was recovered in 119 surgical collections from superovulated donors with a recovery rate of 83.4% (zygotes per corpora lutea). A total of 471 zygotes were microinjected with either the metallothionein human blood clotting factor VIII cDNA (MT-FVIII) or β-lactoglobulin FVIII cDNA (β-Lac VFIII). One to four microinjected zygotes were transferred to 130 recipients of which 76 (58.5%) were diagnosed as pregnant and delivered a total of 124 lambs. Pregnancy rate was significantly increased when four zygotes had been transferred per recipient (70.8% vs. 47.8% to 58.4%). Three transgenic lambs were identified by southern blot analysis (2 males, 1 female). Preliminary culture experiments revealed that microinjection did not impair in vitro development of ovine zygotes. These data indicate that microinjection of FVIII gene constructs into ovine zygotes is compatible with high embryonic survival rates, but the low percentage of transgenic offspring needs further study.