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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CloneEZRPCR 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 a cardiomyocyte-specific transgenic mouse that overexpresses miR-19b and perform phenotypic analysis. Methods: The insertion site of the vector was identified. The corresponding primers were designed according to the recombination principle of CloneEZRPCR Cloning Kit, and the target fragment with the pre-miR-19b precursor sequence (pre-miR-19b) was cloned from the mouse genome , Ligated to the vector with the alpha-MHC promoter using recombinase. The resulting vector was linearized and injected into mouse embryonic stem cells by microinjection. The resulting recombinant embryonic stem cells were transplanted into the uterus of pseudopregnant C57BL / 6J female mice. Specific primers were used to identify the transgenic mice in the resulting offspring, that is, Founder mice, and the corresponding strains were selected for the table Type analysis Results: miR-19b transgenic mice were successfully constructed, and the mean diastolic interventricular septum thickness, diastolic left ventricular free wall thickness and ejection fraction increased in miR-19b transgenic mice. Conclusion: Myocardial specific over-expression of miR-19b can lead to cardiac hypertrophy.