利用CRISPR/Cas9构建敲除小鼠模型研究PPP2R3A基因对心脏功能的影响

To explore the effect of PPP2R3A gene on cardiac function using CRISPR/cas9 knockout mouse model

目的 应用CRISPR/Cas9技术构建蛋白磷酸2调节亚基B″家族α亚型(PPP2R3A)基因敲除小鼠,从分子水平及组织水平上研究PPP2R3A缺失对心脏的影响。方法 将Cas9 mRNA和两个靶向PPP2R3A第3外显子翻译起始密码子附近区域的单导向RNA微注射到C57BL/6小鼠受精卵中。小鼠出生后取其基因组DNA进行聚合酶链反应(PCR)和测序以鉴定基因型,鉴定后,基因PPP2R3A缺失型小鼠为KO组,野生型C57BL/6小鼠为WT组(雄性3只,雌性2只)。小鼠心脏组织经甲醛固定并制成切片后分别进行苏木素-伊红(HE)染色和免疫组织化学染色。提取小鼠心脏组织总RNA和蛋白,应用荧光定量PCR和Western印迹验证基因敲除小鼠的有效性和检测互作蛋白表达。结果 获得F1代PPP2R3A杂合小鼠,PCR和测序结果表明突变小鼠的基因型存在113 bp的缺失突变。与WT组相比,KO组心脏组织中PPP2R3A mRNA和蛋白表达量明显下降(均P<0.05),参与心脏发育的G蛋白信号转导调控因子(RGS)19表达量明显升高(P<0.05)。PPP2R3A蛋白表达受损引起了心脏组织病理学变化。结论 PPP2R3A在体内可能通过与RGS19蛋白互作来参与心脏的发育并对心脏功能产生影响。

Objective To explore the effect of PPP2R3A deletion on the heart at molecular and tissue levels based on the PPP2R3A knockout mice using CRISPR/Cas9 technology.Methods The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at PPP2R3A exon 3 were microinjected into C57BL/6 mouse zygotes.The genomic DNA of the mice was taken after birth for polymerase chain reaction(PCR)to perform sequencing for identifying the genotype.After identification,PPP2R3A knockout mice were in KO group,and wild-type C57BL/6 mice were in WT group(3 males and 2 females).The mouse heart tissue was fixed with formaldehyde and sliced for hematoxylin-eosin(HE)staining and immunohistochemical staining,respectively.The total RNA and protein from the heart tissue of rats were extracted,real-time quantitative PCR and Western blot were used to verify the effectiveness of gene knockout mice and to detect interacting proteins expression.Results F1 generation PPP2R3A heterozygous mice were obtained,PCR and sequencing results showed that the mutant mice genotype had 113 bp deletion mutation.Compared with WT gro up,the mRNA and protein expressions of PPP2R3A in the cardiac tissue were significantly decreased(all P<0.05),the expression of regulator of G-protein signaling(RGS)19,which was involved in heart development,was significantly increased in KO group(P<0.05).The impaired expression of PPP2R3A protein caused pathological changes of heart tissue.Conclusions PPP2R3A might participate in cardiac development and affect cardiac function by interacting with RGS19 protein in vivo.