以精子载体法制备转基因小鼠的初步研究

Study on the Product of Transgenic Mice By Sperm-mediated Gene Transfer

将外源融合基因 BαLA-HI与 DOSPER脂质体等比例混合 ,加入获能精子悬液中 ,37℃、5% CO2 共培养 0 .5h.以这种处理精子作为转基因载体进行小鼠的体外受精及胚胎移植 .在获得的 4 0只移植后代中 ,经 PCR特异片段扩增和 Southern杂交 ,共检测出 2只呈阳性的转基因小鼠 .证明人胰岛素基因已在小鼠染色体上实现了整合 .基因整合率为 5% .

The 1.34 kb DNA fragment containing the encoding region and 3′ flanking region of Human Insulin (HI) gene, and the 0.84kb DNA fragment of the 5′ flanking region in Bovine α lactalbumin (BαLA) gene were obtained by PCR amplification.The 2.2 kb mammary gland specific expression vector carrying HI gene was constructed by gene recombination. The foreign fusion gene were mixed with Dosper liposome according to ratio of 1∶1,laying aside at 37 ℃ for 20 minutes. The semen was collected from the cauda epididymis of sexaully mature proven male mice . The sperm suspention was incubated to capacitation in TYH medium at 37 ℃ in 100% humidity with 5% CO 2 in air for 1.5h.The mixture of liposome and foreign DNA fragment was added into the suspension that had been incubated 1 h.Meanwhile the mature oocytes were recoveried from oviduct of the superovulated female mice. Then the mouse oocytes were fertilized with the sperm that carried the foreign gene .The 2~4cell embryos derived from IVF procedure were transferred into the oviduct or continued to be cultured to the morola stage, then were transferred into the uterine horn of synchronized pseudopregnant recipient mice. Two positive mice were identified among the 40 offspring obtained from embryo transfer by PCR specific fragment amplification and southern blotting hybridization. The results showed HI gene had been integrated on the host chromosomes. While the integration rate was only 5% .