PP2A Cα敲除小鼠心肌细胞模型的构建

Generation of PP2A Cα Knockout Model in Neonatal Mouse Ventricular Myocytes

体外构建PP2A Cα敲除的原代小鼠心室肌细胞模型.选择性地将雄性PP2A Cαfl/fl,Cre/-小鼠与雌性PP2ACαfl/fl,Cre/-小鼠交配.新生小鼠心脏经胰酶消化后差速贴壁获得原代小鼠心室肌细胞,用带有Cre的腺病毒侵染心肌细胞,经荧光显微镜观察和Western-blot检测,分析PP2A Cα的敲除效果.将基因型为PP2A Cαfl/fl,Cre/-的雌雄小鼠进行交配,得到其同样基因型的后代,进而可获得足量基因型为PP2A Cαfl/fl,Cre/-的原代小鼠心肌细胞.用带有重组酶Cre的腺病毒侵染细胞48 h后,可见带有绿色荧光的心肌细胞;Western-blot检测显示,PP2A Cα在Cre腺病毒侵染的心肌细胞可下调60%～80%.本研究成功建立了PP2A Cα敲除的原代小鼠心肌细胞模型.

To generate PP2A Cα knockout model in neonatal mouse ventricular myocytes. Male PP2A Cα^fl/fl- were selectively mated with female PP2A Cα^fl/fl Cre/- mice to obtain abundant offsprings with the same genotype. The neonatal mouse ventricular myocytes were digested by trypsinization and subsequently infected with Cre-adenovirus. The effects of adenovirus infection were observed by fluorescence microscope and identified by western blot analysis. The offsprings in aboundance were obtained by seting up a mating between male and female of PP2A Cα^fl/fl Cre/- mice, as a result, an abundance of neonatal mouse ventricular myocytes with the genotype of PP2A Cα^fl/fl Cre/- were available for Ad-Cre infection. Green fluorescence was observed after GFP labeled Cre-adenovirus（Cre-Ad） infection for 48 h. The expression level of PP2A Cα protein in Cre-Ad infected myocytes was 60% - 80% lower than control. The PP2A Cα knockout model in neonatal mouse ventricular myocytes was successfully generated.