肝细胞特异性表达Cre重组酶转基因小鼠的建立

A transgenic mouse that targets the expression of Cre recombinase in hepatocyte

目的 构建在小鼠肝细胞中特异表达Cre重组酶的转基因小鼠。 方法 利用聚合酶链反应(PCR)获得小鼠肝细胞特异性启动子——白蛋白启动子,指导Cre重组酶在肝细胞中特异表达。通过受精卵显微注射的方法,将转基因载体导入小鼠受精卵中,获得转基因小鼠。利用逆转录聚合酶链反应(RT-PCR)检测转基因小鼠中Cre重组酶转录的组织特异性,并将转基因小鼠与Smad4条件基因打靶小鼠进行杂交,利用PCR和Southern Blot进一步检测Cre重组酶在小鼠体内表达的组织特异性及其介导lox P位点之间发生重组的活性。 结果 获得了白蛋白启动子,构建了转基因载体pAlb-Cre。将转基因载体进行显微注射,共注射了837枚小鼠受精卵。PCR检测显示53只子代小鼠中有7只整合了Cre重组酶基因,Southern杂交结果证实共获得6只基因组上整合Cre重组酶基因的首建者小鼠。RT-PCR结果表明其中3只阳性小鼠的子代鼠在肝脏和睾丸中正确转录了外源基因。将在肝脏和睾丸中正确转录了外源基因的两只阳性鼠与Smad4条件基因打靶的小鼠杂交,通过PCR检测发现Cre转基因与Smad4条件基因打靶双阳性的子代鼠在肝脏中特异表达Cre重组酶,并能介导基因组中loxP序列间的基因重组,此结果通过Southern杂交得到了进一步证实。 结论 成功构建了在小鼠肝细胞中特异表达Cre重组酶?

Objective To construct a mouse that specifically expresses Cre recombinase in hepatocyte. Methods A hepatocyte specific transgenic construct containing mouse albumin promoter, the Cre recombinase gene and the poly (A) of human growth factor gene was generated. The linearized constructs were introduced into the fertilized eggs by microinjection to obtain the transgenic mice. The transcriptional specificity of Cre recombinase was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression and function of Cre recombinase were detected by PCR and Southern Blot after crossing the Alb-Cre transgenic mice with the Smad4 conditional knockout mice. Results The linearized constructs were microinjected into 837 fertilized eggs, and then the 797 effective eggs of microinjected eggs were implanted into the oviducts of 27 pseudo pregnant mice. In the 53 offspring, there were 6 mice carrying the transgene identified by polymerase chain reaction (PCR) and Southern Blot. Cre recombinase transcripts were detected in the livers and testis of the Alb-Cre transgenic mice using RT-PCR. The Cre recombinase was expressed in the livers of the double heterozygous for Alb-Cre and Smad4 floxed allele, and the exon 8 floxed by loxP site was deleted. Conclusion A hepatocyte-specific Cre transgenic mouse was generated successfully. The Cre recombinase expressed specifically in liver and could mediate the recombination between loxP sites in vivo.