FoxO1突变体转基因小鼠的建立

为探究FoxO1对瘦素信号传导的调节机制,本研究构建了重组质粒pEF1α-myc-FoxO1ΔDBD,并将其转染于293Rb细胞,应用Western blotting检测突变体FoxO1ΔDBD的表达;然后采用DNA原核显微注射的方法制备含有突变体FoxO1ΔDBD的转基因小鼠模型,以鼠尾基因组DNA为模板,PCR扩增鉴定基因型;采用实时荧光定量PCR和免疫共沉淀方法检测FoxO1ΔDBD在转录水平和翻译水平的表达,并对FoxO1ΔDBD转基因鼠进行表型分析。结果显示,试验成功构建了重组质粒pEF1α-myc-FoxO1ΔDBD,并在293Rb细胞中检测到了突变体FoxO1ΔDBD的表达,继而获得了10只首建鼠,建立了10个繁育系。试验建立了高G-C含量PCR扩增产物的基因型鉴定方法,并成功地应用于转基因小鼠中。实时荧光定量PCR和免疫共沉淀结果显示,在转录水平和翻译水平上成功地检测出FoxO1ΔDBD的表达,表型分析结果显示,转基因小鼠体重显著低于野生型小鼠（P < 0.05）,提示瘦素的作用增强。综上所述,本试验成功构建了FoxO1ΔDBD转基因小鼠,为探究FoxO1对瘦素信号传导的调节机制提供研究模型。

Establishment of the FoxO1 Mutant Transgenic Mice Model

In order to explore the mechanism of FoxO1 in leptin signal transduction pathway,the recombinant plasmid pEF1α-myc-FoxO1 was constructed and transfected in 293Rb cells to detect its expression by Western blotting.The FoxO1 mutant transgenic mice were constructed by DNA pronucleus microinjection,and total genome was extracted from the mice tail and the genotypes were identified by PCR.The transcription and translation level of the FoxO1ΔDBD were detected by quantitative Real-time PCR and immunoprecipitation,and the phenotypic analysis of the FoxO1 mutant transgenic mice was conducted.The results showed that the recombinant plasmid pEF1α-myc-FoxO1ΔDBD was successfully constructed and expressed in 293Rb cells,and the 10 first generation mice and 10 lines were acquired.The method of genotypic identification of high content of G-C PCR product was established,and the genotypes of transgenic mice were detected successfully.And the expression of mutant FoxO1 was detected successfully on transcription and translation level.The result of phenotypic analysis indicated that the body weights of transgenic mice were significantly lower than wild type mice (P < 0.05),suggesting that the role of leptin was enhanced.In conclusion,the FoxO1 mutant transgenic mice models were successfully established,which provided a model for research the mechanism of FoxO1 in leptin signal transduction pathways.