In Vitro Culture of Bovine Fetal Fibroblast Cells Using Tissue Explant Attachment and Gene Transfection Through Electroporation

The low efficiency and high cost of pronuclear microinjection technology has been the main barrier for transgenic animal production. The production of somatic cell clones by using cultured cells derived from different tissues sheds new lights on transgenic technologies. In order to prepare the nuclear donor cells for bovine transgenic cloning, bovine fetal fibroblast (BFFb) cells were isolated by attaching tissue explants from ear skin of a bovine fetal at 3-4 months gestation stage. The cells grew to confluence 7 days after attachment and were cryopreserved after purification and amplification for 2-3 passages. The cell growth curve was plotted, and the karyotype of the cells within the 10th passages and exceed the 20th passages was analyzed. The plasmid pNEI, which contained the Neo^r gene, the EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode and the human pro-insulin gene regulated by bovine α-lactalbamin promoter for expression specifically in mammary gland, were linearized by digestion with XhoⅠ and purified with phenol chloroform extraction. BFFb cells at passage 3 were harvested at 70% confluence and suspended in HeBES buffer to 5×10⁶ cell/ml. Two hundred microliters of the cell suspension were electroporated with 4 μg linearized pNEI vector in a 2mm Gap cuvette for 1, 5, 10, 15 and 20 ms at 800, 900, 1 000 V/cm, respectively. Cells were checked 24-48 hours after electroporation under fluorescence microscopy for GFP expression, and G418 selection (800 μg/mL) was applied since then. After 2 weeks, selected colonies were counted and maintained in culture medium containing 300 μg/mL G418 for 2-3 passages before cryopreservation. A small part of the cells were analyzed by PCR for gene integration. The data showed that the isolated BFFb cells grew actively, and maintained diploid karyotype even after 20th passages. Bright green fluorescence can be detected from 24 to 48 hours after electroporation, and more colonies were selected at the electroporation condition of 900 V/cm, 5 ms. PCR detection demonstrated that the foreign gene was integrated into the genome. The results indicated that the isolated BFFb cells might be competent for transgenic cloning.