基于Cre-loxP重组酶系统构建肺泡Ⅱ型上皮细胞特异性敲除SENP1基因小鼠

Construction of a mouse model for alveolar type II epithelial cell-specific knockout of SENP1 gene based on the Cre-loxP recombinase system

背景:前期在体外成功构建了SENP1基因沉默的人肺泡上皮细胞系,在细胞水平上研究了SENP1在高氧性肺损伤中的作用。目的:基于Cre-loxP重组酶系统构建肺泡Ⅱ型上皮细胞特异性敲除SENP1基因小鼠模型。方法:将SENP1^(flox/-)小鼠自交得到SENP1^(flox/flox)和SENP1^(flox/-)小鼠;将Sftpc-Cre+/+小鼠与野生型小鼠交配获得更多的Sftpc-Cre^(+/-)小鼠。将Sftpc-Cre+/+或子代Sftpc-Cre^(+/-)小鼠与SENP1^(flox/-)或子代SENP1^(flox/flox)小鼠进行杂交,获得SENP1^(flox/-)Sftpc-Cre^(+/-)双杂合小鼠。将SENP1^(flox/-)Sftpc-Cre^(+/-)小鼠与SENP1^(flox/flox)小鼠杂交,获得SENP1^(flox/flox)Sftpc-Cre^(+/-)小鼠。剪鼠尾提取基因组DNA,行PCR扩增,扩增产物经琼脂糖凝胶电泳确定小鼠基因型。取SENP1^(flox/flox)和SENP1^(flox/flox)Sftpc-Cre^(+/-)小鼠肺组织行免疫荧光双标实验及Western blot以验证SENP1敲除效果;取SENP1^(flox/flox)和SENP1^(flox/flox)Sftpc-Cre^(+/-)小鼠心、肝、肺、肾组织行苏木精-伊红染色以观察两组小鼠各脏器的组织形态。结果与结论:琼脂糖凝胶电泳正确筛选出SENP1^(flox/flox)Sftpc-Cre^(+/-)小鼠。免疫荧光双标实验显示,与SENP1^(flox/flox)小鼠相比,SENP1^(flox/flox)Sftpc-Cre^(+/-)小鼠肺组织中SENP1的平均荧光强度降低(P<0.01),且SENP1和Sftpc未见明显共定位(P<0.01)。Western blot结果显示,与SENP1^(flox/flox)小鼠相比,SENP1^(flox/flox)Sftpc-Cre^(+/-)小鼠肺组织中SENP1蛋白表达降低(P<0.001)。苏木精-伊红染色结果显示SENP1^(flox/flox)和SENP1^(flox/flox)Sftpc-Cre^(+/-)小鼠的心、肝、肺和肾脏组织形态无明显改变。该研究利用Cre-loxP重组酶系统成功构建了肺泡Ⅱ型上皮细胞特异性敲除SENP1基因小鼠,为后续研究SENP1基因在以肺泡Ⅱ型上皮细胞为主要损伤细胞的肺疾病如支气管肺发育不良、特发性肺纤维化中的作用提供了良好的工具。

BACKGROUND:Previously,a SENP1 gene-silenced human alveolar epithelial cell line was successfully constructed in vitro,and the role of SENP1 in hyperoxic lung injury was investigated at the cellular level.OBJECTIVE:To construct a mouse model of alveolar type II epithelial cell-specific knockout of SENP1 gene based on the Cre-loxP recombinase system.METHODS:SENP1^(flox/-)mice were self-crossed to obtain SENP1^(flox/flox) and SENP1^(flox/-)mice;Sftpc-Cre+/+mice were crossed with wild-type mice to obtain more Sftpc-Cre^(+/-)mice.Sftpc-Cre+/+or offspring Sftpc-Cre^(+/-)mice were crossed with SENP1^(flox/-)or offspring SENP1^(flox/flox) mice to obtain SENP1^(flox/-)Sftpc-Cre^(+/-)double heterozygous mice.SENP1^(flox/-)Sftpc-Cre^(+/-)mice were then crossed with SENP1^(flox/flox) mice to obtain SENP1^(flox/flox)Sftpc-Cre^(+/-)mice.The genomic DNA was extracted by tail clipping and amplified by PCR.The amplified product was subjected to agarose gel electrophoresis to determine the mouse genotypes.Lung tissues of SENP1^(flox/flox) and SENP1^(flox/flox)Sftpc-Cre^(+/-)mice were subjected to immunofluorescence double-labelling and western blot assay to verify the knockdown effect of SENP1 gene.Heart,liver,lung and kidney tissues of SENP1^(flox/flox) and SENP1^(flox/flox)Sftpc-Cre^(+/-)mice were stained with hematoxylin-eosin to observe the histomorphology of each organ in the two groups of mice.RESULTS AND CONCLUSION:SENP1^(flox/flox)Sftpc-Cre^(+/-)mice were correctly screened by agarose gel electrophoresis.Immunofluorescence double-labeling experiments showed that the mean fluorescence intensity of SENP1 was reduced in lung tissues of SENP1^(flox/flox)Sftpc-Cre^(+/-)mice compared with that of SENP1^(flox/flox) mice(P<0.01)and no significant co-localization of SENP1 and Sftpc was observed(P<0.01).Western blot results showed that SENP1 protein expression was reduced in lung tissues of SENP1^(flox/flox)Sftpc-Cre^(+/-)mice compared with SENP1^(flox/flox) mice(P<0.001).Hematoxylin-eosin staining showed no significant alterations in the histomorphology of heart,liver,lung and kidney tissues in SENP1^(flox/flox) and SENP1^(flox/flox)Sftpc-Cre^(+/-)mice.This study successfully constructed alveolar type II epithelial cell-specific knockout SENP1 gene mice using the Cre-loxP recombinase system,which provides a good tool for the subsequent study of the role of SENP1 gene in lung diseases such as bronchopulmonary dysplasia and idiopathic pulmonary fibrosis,in which alveolar type II epithelial cells are the main damage cells.