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外源性人TIMP-1基因在转基因小鼠染色体上的整合及定位

ISSN号：1007-7626
期刊名称：《中国生物化学与分子生物学报》
时间：0
分类：Q254[生物学—细胞生物学] Q26[生物学—细胞生物学]
作者机构：[1]中国人民解放军总医院肾病中心暨重点实验室,北京100853, [2]中国科学院遗传与发育生物学研究所,北京100101
相关基金：国家重点基础研究发展规划项目（No.G2000057003）,国家自然科学基金资助项目（No.30000080）,国家自然科学基金创新研究群体项目（No.30121005）基金资助

作者：朱晗玉[1], 陈香美[1], 马润林[2], 张冬芬[2], 任建功[2]

关键词：转基因动物模型, 基质金属蛋白酶组织抑制物-1, 荧光原位杂交, 反向PCR, transgenic mice model, tissue inhibitor of metalloproteinase-1, fluorescence in situ hybridization , inverse PCR

中文摘要：

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Southern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3．8kb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．

英文摘要：

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 hTIMP-1 on transgenic mice chromosomes was cloned by inverse PCR （IPCR）. Further analysis showed that hTIMP-1 was integrated into Chr 17 El. 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.

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