miR-19b转基因小鼠的构建及表型分析

Construction and phenotype analysis of miR-19b transgenic mouse

目的:构建心肌细胞特异性过表达miR-19b的转基因小鼠,并对其进行表型分析。方法:先确定载体插入的酶切位点,根据CloneEZRPCR Cloning Kit重组原理设计相应引物,以小鼠基因组为模板克隆出带有miR-19b前体序列(pre-miR-19b)的目的片段,利用重组酶连接到带有alpha-MHC启动子的载体上。将所得载体线性化后,采用显微注射方法注射到小鼠胚胎干细胞内。再将所得的重组胚胎干细胞移植入假孕的C57BL/6J雌鼠子宫内,在所得的子代,即Founder小鼠中采用特异的引物鉴定出转基因小鼠,并选出相应的品系,进行表型分析。结果:成功构建miR-19b转基因小鼠,且miR-19b转基因小鼠舒张期室间隔厚度、舒张期左室游离壁厚度以及射血分数均有升高。结论:心肌特异性过表达miR-19b可以导致心脏肥大。

Objective：To construct heart-special miR-19b overexpression transgenic mouse and analyze their phenotype. Methods： Firstly,we found out the suitable restriction enzyme cutting site on the plasmid which carrying alpha-MHC promoter,and design the primers according recombination principle provided by CloneEZ~ PCR Cloning Kit. We inserted the pre-miR-19b sequence cloned from mice chromosome into the plasmid using recombinase. Secondly,after linearization of recombinant plasmid, they were injected into the embryonic stem （ES） cells. We transplanted the recombinant ES cells into the uterus of the pseudocyesis female C57BL/6J mice. At last,we selected out the lines of transgenic mouse and analyzed the phenotype of TG mouse. Results：We successfully constructed miR-19b transgenic mouse. The scores of interventricular septal thickness,left ventricle posterior wall and ejection fraction of miR-19b transgenic mouse were increased. Conclusion：The overexpression of heart-special miR-19b may lead to cardiac hypertrophy.