We describe the generation of five sheep transgenic for a fusion of the ovine beta-lactoglobulin gene promotor to the human alpha 1-antitrypsin (h alpha 1AT) genomic sequences. Four of these animals are female and one male. Analysis of the expression of h alpha 1AT in the milk of three of these females shows that all express the human protein at levels greater than 1 gram per liter. In one case initial levels exceeded 60 grams per liter and stabilized at approximately 35 grams per liter as lactation progressed. Human alpha 1AT purified from the milk of these animals appears to be fully N-glycosylated and has a biological activity indistinguishable from human plasma-derived material.

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We have recently described the production of large amounts (< or = 65 grams per litre) of enzymatically active human alpha 1 antitrypsin in the milk of transgenic sheep (Wright et al., 1991). Here, we describe in more detail the expression of the human protein in the milk of these animals throughout the lactation period. Human alpha 1 antitrypsin is also found at much lower levels in the plasma of transgenic ewes before, during and after lactation. It is also detected in male plasma at very low levels. We have previously shown human alpha 1 antitrypsin purified from transgenic sheep milk to be indistinguishable from commercially available human plasma derived alpha 1 antitrypsin in terms of gross sugar content and in vitro activity. Here we extend this comparison to more detailed analyses of glycosylation state, amino-terminal sequence, pI value, and molecular weight determination by mass spectrometry.

The production of transgenic animals from ungulate species is an inefficient and expensive procedure. The development of selection methods to identify the small number of transgenic preimplantation embryos produced following DNA microinjection of one-cell embryos would greatly reduce both the cost and effort of these procedures. This study has examined the fate of the ovine beta-lactoglobulin-human alpha 1-antitrypsin (AATB) minigene construct or a subfragment of this following microinjection into one-cell mouse embryos. It has examined two PCR-based methods that were designed to identify a biochemical difference between microinjected DNA constructs to select preimplantation stage embryos in which chromosomal integration of exogenous DNA has occurred. The two methods involved the modification of the AATB DNA construct either by dam methylation or the substitution of dTTP by dUTP. The dam-sensitive DNA endonuclease DpnI, that was used to digest nonintegrated AATB sequences at sites located between PCR oligonucleotide sequences, was found to interfere with the activity of the subsequent PCR reaction. Analyses of the fate of dUTP-DNA indicated that either repair or replication of microinjected DNA interfered with the ability to distinguish between integrated and nonintegrated DNA constructs in the mid-preimplantation stage embryo. The distribution of microinjected AATB DNA between the blastomeres of individual four and eight-cell stage embryos was also examined by the PCR reaction. Microinjected DNA was not found to be evenly distributed between all the blastomeres of individual embryos.

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