Construction Of Two-gene Co-integrated Into The Mammary Epithelial And Fibroblasts Cell Lines And Genetically Transgenic Rabbit

Growth hormone is a positive regulator of mammary gland development. Dairy animals that are administered growth hormone display enhanced lactation performance, a desirable agricultural trait. The objective of the current research was to generate an improved milk production phenotype in a large animal model using over-expressed GH in the mammary gland to enhance mammary development. While the (3-casein management GH receptor activation and the level of expression amount, it is possible to increase the expression of β-casein promoter.In this study, we use the β-casein promoter and the function gene rhPA to constract the mammary specific expression vector PCL25/rhPA(PtPA). Purpose is get gGH and rhPA double gene integrate the goat fetal fibroblasts and mammary epithelial cells by the method of electric transfection. Analyze the double gene integration goat mammary epithelial cells’s expression of results, as well as two cell donor cells, respectively, as the difference of reconstructed embryos to produce transgenic goat. Research the gGH affects the quantity of rhPA expression and the feasibility of the mammary gland epithelial cells for somatic cell nuclear transfer.First we amplified goat growth hormone gene(gGH) from the plasmid by PCR with the sequence alignment is correct, constructed the mammary gland-specific expression vector PCL25/gGH(PLGH), and use our lab’s plasmid PCL25/rhPA(PtPA) which use β-casein gene as promoter to do the double gene’s integrated experiment. In the co-transfection of goat mammary fibroblasts and epithelial cells, screened double gene integration mammary epithelial cells induced prolactin, the supernatant by ELISA detection of the expression level of rhPA whether was effected by gGH at the cellular level. Using the pronuclear microinjection method for producing a transgenic rabbit, with simultaneous injection of both plasmids PtPA and PLGH, by PCR detection of the rabbit to obtain the integration of the double gene transgenic rabbits, while both donor cells use for preparing transgenic cloned embryos.A total of four transgenic rabbits, which integrates with rhPA and gGH double gene was3, a female rabbit, two of the male. Only one rabbit interates with gGH was the female. A total double gene integration with rhPA and gGH goat mammary epithelial cell lines were15, verified by PCR correct, get double gene integration with rhPA and gGH goat mammary epithelial cell lines were67, verified by PCR correct and5height expression rhPA after ELISA test. Preparation of goat fetal fibroblast donor cells were reconstructed embryos30, goat mammary epithelial cells as donor cells of25reconstructed embryos, the fusion of the former rate of83.33%, which is72.00%, in breast cells for nuclear fusion rate was significantly lower than for the use of fibroblast nucleus. But the difference is small, indicating that the goat mammary epithelial cells can be used for the preparation of somatic cell nuclear transplantation of preparation genetically goats.This study shows that gGH gene can increase the expression level of rhPA which use β-casein as promoter and gGH did not affect the rabbits’growth and development. Through the observation of goat mammary epithelial cells reconstructed embryo nuclear fusion’s situation and found that goat mammary epithelial cells can be used for somatic cell nuclear transfer experiments, but pay attention to the pre-fusion process requires a lot of preparation to ensure the integration of effectiveness.In this study, the double gene transfer successfully prepared two cell lines, and to explore the feasibility of reconstructed embryos,which will lay the foundation for future exploration of multiple exogenous functional gene co-expression in goats, and provide a novel insight into the preparation of unmarked transgenic animals.