CaMKⅡα基因启动子的克隆和神经细胞特异性Cre表达载体的构建

Cloning of the CaMKⅡα promoter and construction of neuron-specific vector with Cre recombinase

目的克隆和鉴定CaMKⅡα基因启动子序列,构建神经细胞特异性Cre重组酶的真核表达载体。方法采用高保真PCR方法分步扩增CaMKⅡα基因5′端8.1kb的调控区DNA片段,该片段包括了CaMKⅡα基因5′端调控区的所有序列。经克隆和部分序列测定后,依次与pCre-IRES-EGFP质粒框架连接,构建载体。结果克隆的CaMKⅡα基因5′端侧翼区酶切图谱与公布的C57BL/6J小鼠相应序列的酶切位点相一致,其中文献报道的富含顺式调控元件的序列与公布的小鼠序列完全相同。CaMKⅡα基因启动子正确地连接于Cre基因的5′端,构建了目标载体。结论这一载体的构建为建立前脑神经细胞特异性表达Cre重组酶的转基因小鼠奠定了基础,有助于神经系统相关疾病的研究。

Objective： To clone and characterize the CaMK Ⅱαpromoter and to construct a neuron - specific vector with Cre recombinase. Methods： 8. 1 kb of CaMK Ⅱα 5′ flanking regulation region was obtained by PCR. This fragment was subcloned , partially sequenced, then reconstructed with pCre - IRES - EGFP plasmid. Results： Restriction endonuclease digestion confirmed that the restriction site of the 8. lkb fragment corresponded with those of the reported sequence from the mouse line C57BL/6J, of which the sequence full of cis - acting elements were the same as those reported. This 8. 1 kb fragment was exactly inserted at upstream from the Cre recombinase. The transgene vector was constructed and named as pCaMK Ⅱα - Cre - IRES - EGFP. Conclusion ： construction of the pCaMK Ⅱα - Cre - IRES - EGFP lays the foundation for the generation of the mice expressing Cre in forebrain neuron, and is helpful to the research of nervous system related diseases.