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目的:建立心肌特异性Creg基因敲除小鼠并初步分析其表型。方法:利用订购的Creg两端插入lox P位点(Cregflox/flox)的小鼠与肌型肌酸激酶特异性启动子驱动的Cre重组酶转基因(Ckmm-cre)小鼠交配,获得Cregflox/+/Ckmm-cre小鼠。再利用Cregflox/+/Ckmm-cre小鼠互相交配,获得基因型为Cregflox/flox/Ckmm-cre的心肌特异性Creg基因条件敲除(Creg conditional knockout,Creg c KO)小鼠。用PCR法进行基因型鉴定。用定量PCR及Western Blot检测心肌组织中Creg表达水平。HE染色观察敲除小鼠与同窝野生型对照小鼠心脏大小及形态。检测两组小鼠心电图。用小动物超声评价两组小鼠左心室收缩功能。结果:1经基因型鉴定,成功获得Creg c KO小鼠。2与野生型对照相比,Creg c KO小鼠心脏中CREG在转录及翻译水平表达降低90%以上。3与野生型对照相比,Cre c KO小鼠的心脏大小、形态、心电图及左心室射血分数均无显著差别。结论:成功建立心肌特异性CREG基因条件敲除小鼠,为进一步研究Creg在心脏疾病中的作用和机制提供了有力的工具。
Objective: To establish cardiac-specific Creg knockout mice and preliminary analysis of its phenotype. Methods: Cregflox / + mice were crossed with Cre-Cre transgenics (Ckmm-cre) mice driven by a muscle creatine kinase-specific promoter using a mouse that had been inserted at the lox P site (Cregflox / flox) / Ckmm-cre mice. Then, Cregflox / + / Ckmm-cre mice were mated with each other to obtain a Creg cO knockout (Creg c KO) mouse of genotype Cregflox / flox / Ckmm-cre. Genotype identification by PCR method. Quantitative PCR and Western Blot were used to detect the expression of Creg in myocardium. HE staining was used to observe the size and shape of heart in knockout mice and wild type control mice. Two groups of mice were tested for electrocardiogram. Left ventricular systolic function was evaluated using small animal ultrasound in two groups of mice. Results: 1 Creg c KO mice were successfully obtained by genotyping. 2 Compared with the wild-type control, CREG expression in the hearts of Creg c KO mice was reduced by more than 90% at the level of transcription and translation. There was no significant difference in cardiac size, morphology, ECG and left ventricular ejection fraction in Cre c KO mice compared with wild-type controls. CONCLUSION: Successful establishment of cardiac-specific CREG knockout mice provides a powerful tool for further investigation of the role and mechanism of Creg in heart disease.