Human Preinsulin Gene Expression Specially In Mammary Gland Of Transgenic Mouse

Human insulin is a kind of hormine which is secreted by pancreas islet 3 cell and it is used mostly in the therapy of the diabetes. A large amount of insulin is require in market, but most of the using insulin is insulin of animal and it can cause immunity. This study is focused on the feasibility of producing human insulin in the animal mammary gland. In this study, we amplified the 1182 bp genome DNA of human proinsulin by pyrobest Taq PCR from human genome. The PCR product was cloned into PGEM-T-Easy DNA plasmid by TA cloning, and hINS gene was analyst and compared to the published sequence ,and found that this hINS gene is same to one of the published sequence and there are four different bases with the other published sequence. The first conversion is lie in intron and the other three conversions are lie in allelic variation .and all these differences maybe caused by the genome from the different kind cf human and the conversion did not cause wrong codonsIn order to validate whether the hINS gene can be translated into the protein ,we constructed an expression casstte which is expressed in the mammary gland of transgenic mouse specially . We amplified the both 6.4Kb upstream and 1.6Kb downstream of bovine a Si-casein gene by LA Taq PCR,and linked the hINS gene between the two streams sequence and be sure that the ture is correctness.We spliced the pronuclei vector and microinject the remain DNA sequence to pronuclei of the mouse zygote.We got 29 mice of G0 generation ,and 8 of the 29 mice are positive result which checked by method of PCR. The 8 mice are cut the tail and extract the genome DNA and then digest them with the Xhol restrict enzyme . The probe is spliced from the PI plasmid and used for Southern-Blot with the dealed DNA ,and 1 of the 8 mice is positive result of the Southern-Blot .The8 transgenic mice lactated naturally .and centrifuge to discard the fatness and the acid. At last ,we got the serum of the milk of 5 transgenic mice and 2 normal mice , checked the human insulin in serum by RlA.The result of the CPM show that two transgenic mice are conspicuous excess to the other transgenic mice and normal mice .and the mount of human insulin achieve respectively to 250uIU/ml ,376 ulU/ml.