The Production of Transgenic IVF Cattle Directed by Goat ß-Casein Promoter/Human Serum Albumin Construct

We have developed a human serum albumin (HSA) mammary gland expression vector which contained the full-long sequences of HAS cDNA and intron 1 as well as goat s-casein gene promoter with the 5’ up-stream regulatory region, exon 1, 2, intron 1 and bovine GH poly A sequences. Direct transfer of this vector into goat mammary gland tissues iudicated that the recombinant construct could transiently express HAS in goat milk. Experiments performed with transgenic mice obtained a high-level of HAS expression in milk produced by three transgenic mice. The HAS contents reached 3.54, 0.21 and 3.03 grams per liter of milk, respectively. In addition, we used 40 cow oocytes for in vitro maturation (IVM) and in vitro fertilization (IVF). We selected 20 zygotes for microinjection of the recombinant construct, followed by culturing the eggs in a favorable condition. 17 embryos developed to a late morula stage. All but one exhibited adequate morphology for biopsy. 16 embryos with god morphology were chosen for biopsy. We aspirated 4 to 6 cells from each of these embryos for preimplantation detection of the transgene using the nested PCR method. We detected specific amplified fragment of HAS gene from embryos. These embryos were then transferred to 8 recipient cows. Three of the cows became pregnant. One cow delivered a male calf. The other two cows suffered miscarriage during midtrimester stage of pregnancy. Tests conducted using Southern analysis indicated that HAS gene has been integrated in the calf’s genome, as predicted by preimplantation detection previously.