MLC_2-糜酶融合基因克隆及转基因小鼠的产生

为研究糜酶基因在体内的结构与功能以及与心肌肥厚的关系，并提高糜酶基因在小鼠心脏中的表达，构建肌球蛋白轻链－２启动子（ｍｙｏｓｉｎｌｉｇｈｔｃｈａｉｎ－２ｐｒｏｍｏｔｅｒ，ＭＬＣ２）－糜酶融合基因并产生转基因小鼠．通过删除糜酶基因启动子序列，构建结构基因克隆，然后与大鼠心脏肌球蛋白轻链－２启动子序列相拼接，构建ＭＬＣ２－糜酶融合基因克隆，回收并纯化融合基因片段，显微注射入小鼠受精卵产生转基因小鼠，经ＰＣＲ扩增、Ｓｏｕｔｈｅｒｎ印迹杂交和ＰＣＲ扩增产物的测序，筛选和确定转基因鼠．在新出生的４６只小鼠中有２只为转基因阳性鼠，且外源基因能稳定遗传给后代，从而获得了可用于研究糜酶基因在体内的结构与功能以及与心肌肥厚的关系的转基因小鼠模型．

The Cloning of MLC 2 Chymase Fusion Gene and the Production of Transgenic Mice

To construct the myosin light chain 2 promoter(MLC 2) chymase fusion gene and produce transgenic mice for overexpression of chymase gene in mouse heart to investigate the structure and function, in vivo ,of chymase gene as well as the relation between chymase and hypertrophy,the MLC 2 chymase fusion gene was constructed through splicing the MLC 2 promoter sequence with the cloned chymase structural gene,whose promoter sequence had been deleted by nuclease BAL31,and cloned through molecular cloning.Purified fusion gene fragments were microinjected into murine eggs and transgenic mice were produced.PCR amplification,Southern blot analysis and sequencing of the products of PCR amplification were used to identify the positive transgenic mice.Two of the forty six survived pups were identified to be the positive transgenic mice harboring the MLC 2 fusion gene,and the transgenic mice could hand down the transgene precisely.The results indicate that a transgenic mouse model for investigating the structure and function, in vivo ,of chymase and the relation of chymase to hypertrophy has been produced.