成纤维细胞特异敲除转录因子ATF3基因的小鼠模型构建

Construction of a mouse model of fibroblast cell-specific activating transcriptiun factor 3 knockout

目的:构建成纤维细胞特异敲除转录因子ATF3基因的小鼠模型。方法:利用胚胎显微注射法构建在ATF3基因携带loxP位点的小鼠(ATF3^(fl/fl)),以成纤维细胞细胞特异性表达的Col1a2-Cre介导ATF3条件性敲除,筛选出成纤维细胞条件性敲除ATF3的小鼠(Col1a2-ERCre:ATF3^(fl/fl));取鼠尾DNA进行PCR鉴定基因型,腹腔注射他莫昔芬诱导该基因敲除;12只敲除小鼠和12只同窝野生型小鼠各分为心脏缺血再灌注(I/R)组和假手术(sham)组,术后2h取心脏组织研磨后流式细胞分选成纤维细胞,进而提取mRNA并反转为cDNA,采用实时荧光定量PCR检测ATF3的表达;另取4只敲除小鼠和4只同窝野生型小鼠复制I/R模型,术后6h取心脏组织进行免疫荧光染色,观察成纤维细胞ATF3表达。结果:鼠尾DNA PCR鉴定基因型结果显示Cre基因型为阳性,ATF3 loxP基因型为纯合阳性;与假手术组相比,野生型小鼠术后心脏成纤维细胞ATF3表达明显升高[(0.02534±0.002654)vs.(0.5982±0.03495),P<0.0001],与术后野生型小鼠相比,术后条件性敲除组心脏成纤维细胞ATF3表达明显降低[(0.5982±0.03495)vs.(0.06456±0.005704),P<0.0001];免疫荧光共定位染色显示野生型小鼠心脏成纤维细胞ATF3蛋白表达,条件性敲除小鼠ATF3蛋白未见表达。结论:成纤维细胞特异敲除ATF3小鼠构建成功,为进一步实验奠定基础。

Objective： To build a mouse model of fibroblast cell-specific ATF3(activating transcription factor 3） conditional knockout. Methods： Using Embryo microinjection method to construct mice expressing ATF3 gene at the loxP site(ATF3^fl/fl）. Col1a2-Cre specific expression of fibroblast cells mediated ATF3 conditional knockout,to get fibroblast-specific ATF3 knockout mouse model Col1a2-ERCre/ATF3^fl/fl. Afterward,the genotype of ATF3 conditional knockout mice was analyzed. Twelve fibroblast cell-specific knockout mice and twelve wild-type littermates were respectively divided into sham group and myocardial ischemia/reperfusion injury(MIRI） group randomly. Flow cytometry sorting cardiac fibroblast,mRNA was extracted and inverted into cDNA,and quantitative real-time polymerase chain reaction(realtime-PCR） were used to determine the expression of ATF3 in cardiac fibroblast. Immunofluorescence staining of heart tissues at 6 h after surgery was used to observe the protein expression of ATF3 in cardiac fibroblast(n = 4）. Results： The genotypes of mice tails DNA were identified by PCR： the genotype of Cre was positive,ATF3 loxP was homozygous positive.Compared with the sham-operated group,the expression of ATF3 in cardiac fibroblasts of wild-type mice was significantly increased in response to ischemia reperfusion [(0. 02534 ± 0. 002654） vs.(0. 5982 ± 0. 03495）,P〈0. 0001）. The expression of ATF3 in cardiac fibroblasts in conditional knockout group after surgury was significantly lower than wild type mice [(0. 5982 ± 0. 03495） vs.(0. 06456 ± 0. 005704）,P〈0. 0001]. Immunofluorescence co-localization staining showed ATF3 protein expression in wild-type mice cardiac fibroblasts,but no expression in conditional knockout mice. Conclusion： The fibroblast-specific knockout of ATF3 mouse was successfully constructed and laid the foundation for further experiments