Detection Of VEGF Transgenic Cashmere Goats

VEGF is a kind of autocrine growth factor of Dermal papilla cells, which can be expressed in primary follicles of humans and animals. Studies have shown that, VEGF is closely related to animal hair follicle development and hair growth. In the transgenic production of cashmere goats, expression and mechanism of action of exogenous genes remain to be detected and explored.In2012, a number of VEGF transgenic cashmere goats by somatic cell cloning technology had been obtained in my laboratory. In this research two transgenic cashmere goats which cashmere production was improved (No.37) and unimproved (No.41) were selected, contrasted with a somatic cell cloned goat (No.5) from the same cell line, to detect the expression of VEGF gene in the transgenic cashmere goats, so that to understand the effects of VEGF on cashmere production.In this experiment, the expression of VEGF on DNA, RNA, protein and individual levels was detected in the three cashmere goats, the results showed:1. Individual cashmere production performance of the three cashmere goats was measured after fleecing, the body/cashmere weight of NO.37, NO.41and NO.5goats was37.5kg/1095g,35kg/460g and36.5kg/450g respectively.2. Preliminary the two transgenic cashmere goats were identified by PCR, and the results showed that NO.37and NO.41cashmere goats both were transgenic positive. Then the results of southern bolt test on those two transgenic cashmere goats were consistented with the results of PCR.3. Realtime PCR testing and western blot detection with GAPDH gene as an internal reference were performed on those two transgenic cashmere goats and the somatic cell cloned goat. The results showed that, compared with the NO.5goat, the RNA and protein expression of VEGF of NO.37goat were3.27and1.82folds in skin, and0.49and1.02folds in blood, repectively. While only0.93and1.16folds in skin and0.24and0.86folds in blood in NO.41goat.4. In this experiment, Glucagon gene as an internal reference, VEGF gene copy number in the two transgenic cashmere goats was detected using absolute quantitative PCR. The standard curve was Log2N (copy number)=-0.3881â–³Ct+0.3093(R2=0.9983, p<0.001), calculated that transgenic copy number in NO.37and NO.41cashmere goats were0.82and0.97respectively. The results showed that both of them exogenous genes were in single copy.5. Skin paraffin slittings of No.37, No.41and No.5goats were prepared and then immunohistochemistry stained. Imaged under the laser scanning confocal microscopy, the fluorescence intensity of the hair follicle and surrounding tissue was measured using the analysis software. The results showed that the fluorescence intensity of VEGF protein in NO.37and NO.41goats was1.21and1.07folds compared to the NO.5goat.6. The skin of the No.37, No.41and No.5goats were treated with frozen sections transversed, then stained by HE. The results showed that the number of primary hair follicles in a hair follicles cluster was not changed in three in transgenic cashmere goats, while the number of secondary follicles varied. The fluff ratio of NO.37, NO.41and NO.5goats was13.78,11.78and9.89respectively.These results indicated that the expression of VEGF in transgenic cashmere goats was specificity in skin tissue, and it high level expression was benefit to improve cashmere production. Meanwhile transgenic animals detecting system have been initially established in this research and will be benefit for understand on transgenic animals.