We have combined gene transfer, by microinjection, with 'in vitro' embryo production technology, enabling us to carry out non-surgical transfer, to recipient cows, of microinjected embryos that have been cultured from immature oocytes. Using this approach, we have established 21 pregnancies from which 19 calves were born. Southern blot analysis proved that in two cases the microinjected DNA had been integrated in the host genome.

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine alpha S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 micrograms ml-1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.

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In the lactating cow, mammary gland-specific hypomethylation occurs at two Hpa II sites in the 5'-flanking region of the alpha S1-casein gene, and one in the 3'-region. These sites, A, B and C, are at nucleotide position -1388, -774 and +18034, respectively, relative to the major transcription start site. Site B was hypomethylated when the alpha S1-casein gene was expressed, and methylated when not expressed. In transgenic mice containing the bovine alpha S1-casein 5' and 3' regulatory elements fused to the human lactoferrin (hLF) cDNA, in some cases similar methylation patterns of sites A and B, as compared to the situation in the cow, were observed. In five mouse lines (out of the seven analysed) expressing the transgene in the milk, site B was hypomethylated in the mammary gland, while it was methylated in liver. In the two other mouse lines, no correlation was found between transgene expression and mammary gland-specific hypomethylation of site B. One of the five mouse lines with transgene expression and showing mammary-gland-specific hypomethylation of site B was studied in detail. In this mouse line, induction of transgene expression preceded hypomethylation of site B.

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