Abstract: In order to prepare bovine mammary gland bioreactor for production of human recombinant GDNF, the female bovine fetal fibroblast cells were successfully isolated using tissue bulk attachment. The fibroblast cells were cultured consecutively for 75 days for further morphologic observation and chromosome analyzing. The plasmid vector pNR-GDNF, which contained the Neo^r gene and the DsRed2 gene as positive selection marker genes and human GDNF cDNA gene regulated by bovine beta-casein promoter for specific expression in mammary gland, was transfected into the bovine fetal fibroblast cells by electroporation. After selection with G418 for 7 days, resistant cells expressing red fluorescence protein were isolated, cultured, expanded and cryopreserved by standard procedures. The transgenic cells were indentified by PCR. The results showed that bovine fetal fibroblast cells possessed normal morphology, multiplication characteristics and chromosome number and the foreign gene was integrated into the genome.

Key words: Bovine fetal fibroblast, Isolation and culture, Gene transfer, GDNF