The Study Of Target Gene Copy Number/Expression Level And Production Performance In Tβ4Transgenic Cashmere Goats

Since the21st century, with the continuous improvement of genetic engineering, microinjection, somatic cell cloning, embryo transfer and other related technologies, transgenic animals has been rapid developed. In2008, our country launched a major project to cultivate new varieties of transgenic animals. Now a variety of transgenic animals have been successfully produced, therefore, the study of transgenic animals has made significant achievement. Our research team in2011also produced thymosin β4transgenic cashmere goats. Studies have shown that:Overexpression of thymosin β4can promote the growth and development of hair follicles; copy number of exogenous gene are associated with exogenous gene expression level. Therefore, That production performance of Tβ34transgenic cashmere goats was improved significantly and not significantly, was firstly used to analyze the relationship between copy number and the expression of exogenous gene, then by comparison with somatic cell cloning cashmere goat, to study inherent link between expression level of Tβ4gene and production performance of cashmere goats, so that these studies can provide reliable technical methods and theoretical basis for the detection of transgenic cashmere goats and the cultivation of GMO new varieties.1. Detection of positive transgenic cashmere goats The first, PCR and Southern blot were simultaneously used to detect positive transgenic cashmere goats, then using PCR identify exogenous gene constructs. The results showed that:four cashmere goats detected are all positive, and multiple sites of exogenous gene (Kap6.1, CMV promoter, DsRed and neomycin gene) integrated into the transgenic cashmere goats genome.2. Detection of exogenous gene copy numberThe cashmere goat endogenous gene glucagon served as an internal control to calculate exogenous gene copy number. Using real-time quantitative PCR, the standard curve between copy number and△Ct was established. Standard curve was: log5N=-0.3794△Ct+0.8384(R2=0.9969) and copy number of NO.28、NO.91、 NO.14and NO.94was2、4、6and1respectively.3. The relative expression of Tβ4geneIn this experiment, GAPDH was used as internal reference, the relative expression of Tβ4gene mRNA was calculated. Drawing standard curve and melting curve, the correlation coefficient of standard curve was0.99957and0.99726, amplification efficiency was0.97and0.96respectively. The relative expression of Tβ4gene mRNA in NO.28、N0.91、NO.14and NO.94goats was2.68,2.46.2.36and2.57fold compared with No.81goat, β-actin used as internal reference, the relative expression of Tβ4gene protein in NO.28、NO.91、NO.14and NO.94goats was1.384.1.380.1.166and0.982fold compared with No.81goat.4. The expression of Tβ4gene in hair follicle, P/S and cashmere productionTβ4gene expression level in hair follicle was analyzed by paraffin section and immunohistochemical technology, the results showed that the expression of Tβ4gene in NO.28、NO.91、NO.14and NO.94goats was1.908、1.442、1.233、1.331fold compared with No.81goat. P/S of cashmere goat in skin tissue was be measured by frozen section and HE staining, the results showed that in NO.28、NO.91、NO.14、 NO.94and NO.81goats, P/S was14.167、14、12、10and11; cashmere production was1150、1140.805.680and740g, respectively.Based on the above research, we get overexpression of Tβ4gene in transgenic cashmere goats may promote growth and development of hair follicles, and improve the production of cashmere goat and P/S in hair follicles.