外源性人TIMP-1基因在转基因小鼠染色体上的整合及定位

为探讨外源基因人基质金属蛋白酶组织抑制物-1（human tissue inhibitor of metalloproteinase-1, hTIMP-1）基因在转基因小鼠家系染色体上的整合和精确定位，应用Southrn印迹检测外源基因在染色体上整合的位点及拷贝数.结果表明，外源基因是以单拷贝、单位点形式整合；应用荧光原位杂交（fluorescence in situ hybridization, FISH）技术检测F4～F20代转基因小鼠中外源基因的整合.结果证明，该家系转基因小鼠自F4代起是纯合子，外源基因整合在17号染色体E区；反向PCR法（Inverse PCR, IPCR）克隆出约3.8 kb外源基因整合位点处的侧翼序列.分析表明，外源基因整合在17号染色体E1.3区，ALK（anaplastic lymphoma kinase, ALK）基因第23个内含子区域.结果提示，获得的转基因小鼠为纯系，外源基因hTIMP-1已稳定整合在转基因小鼠染色体上，并能遗传给后代.

Integrationand Location of Human TIMP-1(hTIMP-1) Geneon Chromosomes of Transgenic Mice

To study the integration and exact location of human tissue inhibitor of metalloproteinase-1((hTIMP-1)) gene on chromosomes of transgenic mice line,the numbers of integration sites and copies of hTIMP-1 were detected by Southern blotting.The result confirmed that the foreign gene was integrated into the transgenic mice chromosomes in a manner of single copy and single locus.Then fluorescence in situ hybridization(FISH) was used to determine the integration of hTIMP-1 on chromosomes of transgenic mice from F4 to F20 generation,which showed that this transgenic mice line was homozygous from F4 and the hTIMP-1 gene was integrated into Chr 17 E.The 3.8 kb flanking sequence of integration site of hTIMP1 on transgenic mice chromosomes was cloned by inverse PCR(IPCR).Further analysis showed that hTIMP-1 was integrated into Chr 17 E1.3 on the 23rd intron region of anaplastic lymphoma kinase gene with 20 kb being eliminated in this intron.These results indicated that the transgenic mice line was homozygous,with hTIMP-1 integrated stably into the chromosomes,and could be transmitted from founder to offsprings.