Establishment and Characterization of SV40 T Gene-Transformed Dairy Goat Mammary Gland Epithelial Cell Lines
  Revised:May 26, 2003
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KeyWord:Simian virus 40, Genes,Goat,Breast,Epithelial cell,Biological characteristics
SONG Hong-qin,SUN Huai-chuang,XU Yi-min,WANG Bao-an
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Abstract:
      Objective To establish goat mammary gland epithelial cell lines for evaluation of mammary gland-specific expression components.Methods A pair of primers were synthesized according to the previously published DNA sequence and used to amplify the SV40 T gene from COS-1 cells by high fidelity PCR.The SV40 T gene was subcloned into eukaryotic expression vector pTarget with its neo gene deleted and the resulting recombinant vector pTarget-LT was transfected into primary goat mammary gland epithelial cells.The transformed cell clones were obtained by limited dilution and repeated passage.Results An expected PCR product of 2.5?kb was obtained,which was 99% identical in sequence to the previously published SV40 T gene.Following transfection and repeated in vitro passage,49 clones were obtained with a clone forming rate of 26.7% and a logarithmic growth period at the forth day post-plating.Dot blotting analysis showed that the SV40 T gene was integrated into the transformed cell genome.The transformed cells had a normal karyotype with no tumorigenicity to nude mice and were not able to grow in soft agar culture.One of the transformed cell clone had been passed for more than 30 generations,which maintained normal morphology of mammary gland epithelial cells and was able to form glandulous structures on collagen matrix.Conclusion These results showed that the transformed mammary gland epithelial cell line shared similar biological characteristics of other transformed cell lines.
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