CCN1基因肺特异性条件敲除小鼠模型的构建和鉴定

Generation and evaluation of CCN1 gene knockout mice by Cre-LoxP technology

目的:构建Cre重组酶系统调控的肺特异性的富半胱氨酸血管生成诱导因子61(CYR61,又称CCN1)基因条件敲除小鼠,为肺组织内CCN1在炎症中扮演的角色和作用机制的体内实验研究提供更特异的动物模型。方法:将引进的CCN1^(flox/flox)转基因小鼠和肺上皮细胞特异性表达Cre重组酶工具鼠分别进行繁殖和鉴定,然后将2种小鼠进行杂交并对其子代小鼠的基因型进行鉴定,其子代基因型为Cre^(+/-)CCN1^(flox/flox)的小鼠为本研究所构建的肺上皮细胞特异性CCN1基因条件敲除小鼠。结果:Cre^(+/-)CCN1^(flox/flox)的小鼠被成功繁殖并鉴定。使用他莫昔芬腹腔注射处理Cre^(+/-)CCN1^(flox/flox)实验组及Cre^(-/-)CCN1^(flox/flox)对照组,分别使用RT-PCR、免疫荧光、免疫组织化学、Western blot检测肺组织中CCN1的mRNA和蛋白表达,与对照组比可见实验组肺组织中CCN1的mRNA和蛋白表达均明显下降。结论:本研究基于Cre-Lox P技术成功构建了肺上皮细胞特异性CCN1基因条件敲除小鼠,并能稳定传代,可为进一步研究CCN1在肺组织内炎症中调控作用和机制奠定了动物模型的基础。

Objective： To establish and identify the cysteine-rich 61 （CYR61/CCN1） gene knockout mice via Cre-LoxP conditional gene knockout technology, and to build animal model for researchers to explore the role and mechanism of CCN1 gene in lung infammation. Methods： The introduced CCN1fox/fox mice and the mice that express pulmonary epithelial cell-specifc Cre recombinase were bred and authenticated separately. After successful identifcation, two kinds of mice were hybridized. The off-spring mice with Cre^＋/-CCN1fox/fox geno-typing were the objective mice. Results： Cre^＋/-CCN1fox/fox mice were successfully bred and identifed. An in-traperitoneal injection of tamoxifen was administered to stimulate the experiment group （Cre^＋/-CCN1fox/fox） and the control group （Cre^-/-CCN1fox/fox）. RT-PCR, immunofuorescence, immunohistochemical and Western-blot were applied to detect mRNA and protein expression of CCN1 in lung tissue. There was a signifcant decrease of CCN1 mRNA and protein expression in the lung tissues of experiment group compared with the control group. Conclusion： Based on the Cre-LoxP conditional gene knockout technology, we succeed in building a group of lung epithelial cell-specifc CCN1 gene knockout mice which can stably go down to the future generation. This kind of mice would supply a technical basis for animal models in further researches of the regulation and mecha-nism of CCN1 gene in the infammation of lung tissues.