A Goat BLG/CMV-bast Vector Targets Human-lactoferrin CDNA Expression In Cells And The Mammary Gland Of Transgenic Mice

A variety of transgenic animal species are being used to produce biopharmaceutical proteins. The period that the livestock animals mammary gland bioreactor studies is long, the cost is expensive, the risk is great, so it is necessary to detect expression carrier rationality and validity before producing the transgenic livestock animals. In this study, these detecting methods which include cell culture and transgenic mice were used to detect the mammary expression vector (BLC14).1. The construction of expression vector(BLC14): The gene elements including the goat BLG regulatory sequences containing the 4.2 kb 5`end and 1.8 kb 3`end regulatory regions, the hLF cDNA, the enhancer sequences of human Cytomegalovirus(CMV), SV40 polyA sequences and neo-resistant gene were used to construct into the mammary gland special expression vector——BLC14.2.The culture of goat mammary gland epithelial cells(GMECs): Primary goat mammary cells were derived from biopsy tissue obtained from healthy lactating dairy goat, the tissue was digested by collegenase I and hyaluronidase and the cells were collected, then the purified mammary epithelial cells were obtained by trypsin digestion in medium DMEM/F12 through 2-3 passages. The purified mammary epithelial cells were transfected with BLC14 gene fragments by electroporation. After 59 colonies of BLC-14 were obtained by selection of G418 for about 14d, positive clone cells were proliferated and induced by hormone. The result of ELISA on cultured cells supernatant showed that 8 colonies expressed the exogenic gene and secreted hLF protein, the rate of expressed clones was 13.6%.3.The generation of transgenic mice: 1486 eggs of C57♀×ICR♂were microinjected with the 10.6 kb fragment digested from the expression vector with SalI and NotI. 683 eggs were transferred into 16 forster mothers, 10 of which were pregnant and 46 pups were obtained, among which 4 pups (1♀, 3♂) were transgenic mice, the rate of integration was 8.69%. 6 pups of No. 25(♂) were transgenic mice, the rate of integration was 37.5% (6/16); but no pups of No. 11(♂) were transgenic mice.The result of ELISA assay and Western blot assay showed that the milk of the G0 transgenic mouse (NO.19) expressed rhLF. The level of rhLF expression was about 1.5mg/mL. Western blot result indicated that the molecular weight of rhLF was about 80 KD.This study showed that the expression vector-BLC14 can be expressed by the mammary gland epithelial cells and in the mammary gland of the transgenic mouse. The results indicated that the expression vector could be used for producing the transgenic livestock animals whose mammary gland could express rhLF.