312 CONSTRUCTION OF SPECIFICALLY EXPRESSED VECTOR IN MAMMARY GLAND FOR lacS AND ITS TRANSFECTION INTO BOVINE FETAL FIBROBLASTS MEDIATED BY LIPOSOME

Abstract

This study was designed to optimize conditions for transfection of a mammary gland specific transgene into bovine fetal fibroblasts. Transfection of Sulfolobus solfataricus β-glycosidase gene (lacS) was mediated by liposome. Neomycin resistance (Neo^r) and enhanced green fluorescent protein (EGFP) gene were used as genetic markers to screen transgenic somatic cells. A 0.92-kb fragment of bovine β-lactoglobulin gene sequence was obtained from bovine genome by PCR amplification and was inserted into the T site of pMD19-Simple T plasmid. 1.49 kb of lacS gene coding sequence was cloned from Sulfolobus solfataricus genome by PCR amplification and inserted into the pUC19 plasmid. The coding sequence of Neo^r was derived by PCR amplification from pIRES2-EGFP plasmid and inserted into the BamHI/NheI site of pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a Neo^r and an EGFP gene, which were linked by an internal ribosome entry site sequence downstream of the cytomegalovirus (CMV) promoter. Finally, the vector pNIE was assembled into the pUC19 plasmid, thus creating a pBLI vector, which contained the Neo^r and EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode and the lacS gene regulated by bovine β-lactoglobulin promoter for specific expression in mammary gland. Bovine fetal fibroblasts (bFF) were isolated from the ear skin of female fetuses at the age of 2 to 3 months. The cells proliferated well and grew normally in culture, with typical fibroblast morphology and growth curve. The effects of different concentrations of transfection and pBLI were compared on the efficiency of transfection. The passage 4 bFF cells at 70 to 80% confluency were transfected in a 24-well culture plate. 2 × 10⁵ cells were cultured in DMEM with 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 μg of pBLI using transfection (1, 2, 3, 4, 5, and 6 μL) for 48 h, respectively. The transfected cells were cultured for 48 h before adding G418 at concentrations of 200, 300, 400, 500, 600, 700, 800, and 900 μg mL^–1 for 14 d, respectively. Positive cell colonies were selected and purified through both the expression of Neo^r and EGFP gene under a fluorescence microscopy. The selected colonies were propagated in DMEM containing 300 μg mL^–1 G418. The results showed that bright green fluorescence could be detected at 48 h after transfection. 1.0 μg of pBLI plasmid and 3 μL of transfection yielded the desirable efficiency of transfection. More transgenic bFF colonies were selected by G418 at the concentration of 800 μg mL^–1. In conclusion, a specifically expressed vector in mammary gland for lacS gene was successfully constructed, transfection parameters were developed, and efficient screening measures were established for detecting transgenic somatic cells.