利用Cre-loxP系统构建AT2细胞特异性敲除HDAC3基因小鼠

Establishment of Mouse Model with AT2-specific HDAC3 Gene Knockout by Cre-loxP System

目的应用Cre-loxP重组酶系统构建AT2细胞特异性敲除HDAC3基因小鼠HDAC3^(flox/flox)Sftpc-Cre(+/-)模型。方法首先将Sftpc-Cre(+/-)小鼠自交以获得更多的Sftpc-Cre(+/-)小鼠,将HDAC3^(flox/-)小鼠自交以获得HDAC3^(flox/flox)小鼠和HDAC3^(flox/-)小鼠;然后,将Sftpc-Cre(+/-)小鼠与HDAC3^(flox/flox)小鼠或HDAC3^(flox/-)小鼠杂交,得到双杂合HDAC3^(flox/-)Sftpc-Cre(+/-)小鼠;最后将HDAC3^(flox/-)Sftpc-Cre(+/-)小鼠与HDAC3^(flox/flox)小鼠或HDAC3^(flox/-)小鼠杂交,获得目的小鼠HDAC3^(flox/flox)Sftpc-Cre(+/-);对产生的子代剪取鼠尾提取基因组DNA,选取相对应的引物对目的基因进行扩增,扩增产物经琼脂糖凝胶电泳实验以鉴定基因型。选取6~8周龄的目的小鼠及对照组小鼠(HDAC3^(flox/flox))各3只,取肺组织进行免疫荧光双标实验以验证HDAC3敲除效果,取心脏、肝脏、肺、肾脏组织进行HE染色以观察两种小鼠各脏器的组织形态。结果琼脂糖凝胶电泳实验正确鉴定出了子代小鼠,筛选出了目的小鼠HDAC3^(flox/flox)Sftpc-Cre(+/-)。HDAC3在小鼠肺AT2细胞中被成功敲除。两种小鼠的心脏、肝脏、肺和肾脏组织形态无明显改变。结论本研究利用Cre-loxP技术成功构建了AT2细胞特异性敲除HDAC3基因小鼠,为后续进一步研究HDAC3基因在肺部疾病发生、发展中的作用提供了优良的工具。

Objective To construct HDAC3^(flox/flox)Sftpc-Cre(+/-)model of AT2 cell specific knockout HDAC3 gene mice by Cre-loxp recombinase system.Methods First,Sftpc-Cre(+/-)mice were selfed to obtain more Sftpc-Cre(+/-)mice,and HDAC3^(flox/-)mice were selfed to obtain HDAC3^(flox/flox)mice and HDAC3^(flox/-)mice;then,Sftpc-Cre(+/-)mice were hybridized with HDAC3^(flox/flox)mice or HDAC3^(flox/-)mice to obtain double heterozygous HDAC3^(flox/-)Sftpc-Cre(+/-)mice;Finally,HDAC3^(flox/-)Sftpc-Cre(+/-)mice were hybridized with HDAC3^(flox/flox)mice or HDAC3^(flox/-)mice to obtain the target mice HDAC3^(flox/flox)Sftpc-Cre(+/-).The genomic DNA of the offspring was extracted from the mice tail,and the corresponding primers were selected to amplify the target genes.The amplified products were tested by agarose gel electrophoresis to identify the genotype.Three target mice with HDAC3^(flox/flox)Sftpc-Cre(+/-)aged 6-8 weeks and three control mice with HDAC3^(flox/flox)were selected.The lung tissues were taken for immunofluorescence double labeling experiment to verify the effect of HDAC3 knockout,and the heart,liver,lung and kidney tissues were stained with hematoxylin-eosin to observe the tissue morphology of each organ of the two kinds of mice.Results The progenitor mice with HDAC3^(flox/flox)Sftpc-Cre(+/-)were correctly screened out by agarose gel electrophoresis.HDAC3 was successfully knocked out from AT2 cells in the lung of mice.There were no significant changes in the morphology of heart,liver,lung and kidney of the two kinds of mice.Conclusion This study successfully constructed AT2 cell specific knockout HDAC3 gene mice by Cre-loxP technology,which provides an excellent tool for further study of the role of HDAC3 gene in the occurrence and development of lung diseases.