The Study On Expression And Inheritance Stability Of Exogenous Gene VEGF In Tansgenic Cashmere Goats

VEGF (vascular endothelial cell growth factor), which expressed in hair follicle of animals, is a kind of autocrine growth factor of dermal papilla cells. Results of studies have shown that, expression of VEGF gene can induce in vivo angiogenesis and has closely related to animal hair follicle development and hair growth. The VEGF gene had been chosen as exogenous gene for transgenic breeding of Arbas Cashmere Goats in our laboratory, so that to obtain high cashmere yield transgenic cashmere goats, and to provide experimental basis for further exploring the mechanism of exogenous VEGF gene in animal body.In recent years, transgenic VEGF gene cashmere goats have been obtained by somatic cell cloning technology in our laboratory, including significant improved in cashmere yield ones and no significant improved in cashmere yield ones, and adult transgenic cashmere goats were planned breeding to produce offspring goats. In this study, a transgenic cashmere goat (No.1) with significantly improved yield and its two sub generation transgenic cashmere goats (No.2, No.3), and a transgenic cashmere goat (No.4) without significantly improved yield and its two sub generation transgenic cashmere goats (No.5,No.6) were chosen for testing, and a somatic clone cashmere goat (No.7) from the same cell line was compared. The genetic efficiency and expression of exogenous VEGF gene in different generations of transgenic cashmere goats were observed, so that to provide experimental evidence for further study on the heredity of exogenous gene in transgenic animals.In this experiment, the samples were detected on DNA, RNA and protein three levels:(1)Identified by PCR, No.1, No.2, No.3, No.4 goats were genetically modified positive. No.6 and No.5 goats were genetically modified negative.(2) Using the Glucagon gene as an internal standard, the copy number of VEGF gene was detected by absolute quantitative PCR method. The standard curve formula: log2N=-0.3879ACt+0.3122(R2=0.9978, p<0.001) The exogenous gene copy number of the transgenic cashmere goats were: No.1,1.235; No.2,0.977; No.3,0.962; No.4,1.884. The results indicated that No.1, No.2, No.3 goats were exogenous gene single copy, No.4 goat was double copy.(3) Using GAPDH gene as reference, the expression of exogenous gene was detected by Real-time-PCR and Western blot. The results showed that compared with somatic cells clone goat No.7, the expression of mRNA and protein in skin tissues of transgenic cashmere goats were:No.1,3.53 and 2.93 times. No.2,2.78 and 2.56 times.No.3,0.95 and 1.24 times. No.4,1.23 and 1.03 times. No.5,0.78 and 0.96 times. No.6,1.02 and 0.72 times. The expression of mRNA and protein in blood of transgenic cashmere goats were:No.1,0.57 and 0.98 times. No.2,0.78 and 0.87 times. No.3,0.69 and 1.12 times.No.4,0.32 and 0.94 times. No.5,0.91 and 1.12 times. No.6,0.89 and 0.83 times.The results showed that the expression of exogenous VEGF gene in transgenic cashmere goats had skin tissue specificity, and some exogenous gene in transgenic cashmere goats could be inherited to the next generation. There was no direct relationship between the expression of exogenous gene or the genetic efficiency and copy number. The results of this research will be benefit.Further research on the heredity of exogenous gene in transgenic animals.