| The production of recombinant protein is one of the major successes of biotechnology, and animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactors are being used for this purpose. However, whether the transgenic domestic animals are able to efficiently express the recombinant protein in their mammary gland is difficult to be validated before birth of the transgenic offspring.In this study, a simple and efficient method was established to evaluate the functionality of animal mammary gland tissue-expressed cassettes. The gene transfer vector pGBC2LF was constructed firstly, and the expression of human lactoferrin (LF) gene cDNA was regulated by the goat beta-casein gene 5' flanking sequence. Subsequently, the influence of the DNA concentration, the DMSO concentration, and the proportion of the linear and circular DNA for associating DNA with spermatozoa were evaluated to obtain the most efficient transfection. The results showed that, rabbit offspring were produced with DMSO-sperm mediated gene transfer (DMSO-SMGT), and 46 rabbits (57.1%) of all 89 rabbits were positive transgenic ones. Seventeen of 21 (81%) transgenic female rabbits, as mammary gland bioreactor models, could express the human LF protein in their glands. It was demonstrated that transgenic rabbits could be produced by DMSO-SMGT and human lactoferrin protein could be tissue-expressed.Nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and 64.8% (in vivo mature oocyte) and 51.7% (in vitro mature oocyte) reconstructed embryos could develop to blastocyst in vitro.
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