CaMK_基因启动子的克隆和神经细胞特异性Cre表达载体的构建.pdfVIP

∃ 42∃ 2008 16 3 CaMK C re , , , (福建医科大学基础医学院细胞生物学与遗传学系, 福州 350004) : 目的 克隆和鉴定 CaMK 基因启动子序列, 建神经细胞特异性 C re重组酶的真核表达载体方法 采用 高保真 PCR 方法分步扩增 CaMK 基因 5 端 8. 1kb的调控区 DNA片段, 该片段包括了 CaMK 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 pC re- IRES- EGFP质粒框架连接, 建载体结果 克隆的 CaMK 基因 5 端 侧翼区酶切图谱与公布的 C5 BL /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CaMK 基因启动子正确地连接于 C re基因的 5 端, 建了目标载体结论 这一载体的 建为建立 前脑神经细胞特异性表达 C re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 : CaMK 基因启动子; 克隆; C re重组酶; 表达载体; 神经细胞特异性 : R3943 : A : 1006- 9534 ( 2008) 03- 0042- 03 C lon ing o f the CaMK p romo te r and const ruct oi n o f neuron- spec ific vectorw ith C re recomb inase. X U Ying, ZH OU Chang -w en, LIN Zhao - hua, KE Qi. (Fuj ian M edica l Uni ersity, Fuzhou 350004) Abst ract: Objective: To clone and characterize the C aMK prom oter and to construct a neuron - specific vector w ith C re recomb inase. M ethods: 81kb ofC aMK 5 flank ing regu lation regionw as obtained by PCR. This fragmentw as subcloned , partial ly sequenced, then reconstructedw ith pCre- IRES- EGFP plasm id. Results: Restriction endonuclease digestion confirm ed that the restriction s ite of the 81kb fragm en t correspondedw ith those of the reported sequence from themouse line C5 BL/6J, of wh ich the se quence fu ll of cis- acting elem entsw ere the sam e as those reported. This 81kb fragm ent was exactly inserted at upstream from the Cre recomb inase. The transgene vectorw as constructed and nam ed as pCaMK - Cre- IRES- EGFP. Conclusion: construction of the pCaMK - Cre- IRES- EGFP lays the foundation for the generation of them ice express ing C re in forebrain neuron, and is help fu l to the research of nervous system related d iseases. Key w ords: Prom oter; C lone; Cre recomb inase; Transgene construct;