GLUT4-Cre转基因模型建立与Cre酶特异性表达

目的：建立骨骼肌和脂肪组织特异性表达Cre重组酶基因的转基因小鼠，以期在上述组织细胞中实现相关基因的条件性剔除。方法：选择人葡萄糖转运蛋白4(GLUT4)基因的启动子驱动Cre重组酶的特异性表达，利用基因重组技术，构建GLUT4-Cre转基因表达质粒。通过显微注射法将该质粒导入小鼠受精卵细胞核，获得的新生鼠经PCR和Southern杂交方法进行基因型的分析鉴定，常规方法培育、传代转基因鼠。用RT-PCR和免疫组化法对Cre酶在阳性转基因鼠体内mRNA和蛋白表达水平进行检测。结果：获得5只阳性转基因首建鼠，其中3只已建系并稳定传代。在脑、脂肪组织中检测到Cre基因表达，而心肌、骨骼肌及肝脏组织中未检出转基因表达。结论：成功构建了在脑、脂肪组织中特异性表达Cre的转基因鼠，为研究与2型糖尿病、胰岛素抵抗等相关的条件性基因剔除小鼠模型的建立奠定了基础。

Construction and tissue specific expression of Cre transgenic mice

Objective To generate transgenic mouse model in which Cre enzyme expresses in a tissue specific manner. Methods The promoter of human glucose transporter type 4 (GLUT4) gene was cloned and used to drive Cre expression. Through microinjection, linearized Cre expression vector was injected into the pronuclei of zygotes of C57BL/6J and CBA mice. The injected zygotes were implanted into the oviducts of pseudopregnant female mice. The integration of exogenous genes in the offsprings was detected by PCR and southern blot analysis. Expression of Cre in tissues of the adult mice was evaluated by RT-PCR and immunohistochemical analysis. Results Five founder mice were obtained, which contained Cre genes in their genomes. Three of them had offsprings and formed transgenic mouse lines. In the line 8 the expression of Cre gene was detected in the brain and adipose tissues, but not detected in the liver and skeletal muscle. Conclusion The transgenic mouse model in which Cre gene is tissue-specifically expressed has been generated. This model will benefit the generation of conditional knock-out mouse models with genes which might be involved in type 2 diabetes or insulin resistance.