Targeting the human lysozyme gene on bovine αs1- casein gene locus in fibroblasts

Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine as1-casein gene locus. In this case, the expression of human lysozyme could be regulated by the endogenous cis-element of as1casein gene in bovine mammary glands. In order to analyze the bioactivity of the vector, the targeting vector was stably transfected and randomly integrated into mouse mammary epithelial cells. Reversetranscription- polymerase chain reaction (RT-PCR) and western blot results showed that, the bovine as1-casein promoter in the 5' arm was able to direct the efficient expression and secretion of human lysozyme in mammary epithelial cells. Turbidimetric assay showed that the antibacterial activity of lysozyme in transfected cells culture medium was 180 U/ ml. To obtain the gene targeted cells line, bovine fetal fibroblasts were isolated and transfected with linear targeting vector (21.9 kb) using nucleofector device, which the transfection rate was about 25%. After seven rounds of independent cell transfection, a total of 8 x 10 7 cells were transfected, 118 colonies were expanded and analyzed by PCR, but none were found to be targeted. However, the targeted events were detected in the mixed cells which did not formed obvious colonies in five 10 cm dishes. Thus, these results indicate that, the 8.2 kb exogenous genes could be site specifically integrated into the transcriptionally silent as1-casein gene locus in fibroblasts, but the unfavorable chromatin structure in such loci may have a disadvantage to targeted colonies formation in expansion stage. We suggest that minimizing the length of in vitro culture time and relax selection as soon as colonies become evident might prevent such loss of targeted cells.