脑源性神经营养因子(rBDNF)转基因小鼠载体的构建和体外表达

Establishing and overexpressing rat brain-drived neurotrophic factor conditional transgenic vector in vitro

目的构建大鼠脑源性神经营养因子(rBDNF)的条件性转基因小鼠载体,鉴定其在体外细胞系中的表达。方法用PCR方法扩增rBDNF片断,插入pNAO载体(lox-Stop-lox-IRES-EGFP),构建成rBDNF条件性基因载体(lox-Stop-lox-rBDNF-IRES-EGFP),使用Fugene 6转染试剂,将其同Cre重组酶表达质粒pGK-Cre共同导入HEK-293细胞系中,通过荧光显微镜观察、PCR及ELISA方法确定Cre-lox同源重组。结果经酶切和测序鉴定,rBDNF条件性基因载体构建成功。共转染后的HEK-293细胞中,观察到绿色荧光蛋白(EGFP)的强荧光表达;细胞基因组DNA中检测到Cre-lox基因重组;对rBDNF定量检测的ELISA结果显示,细胞内有大量的rBDNF释放到细胞外。结论rBDNF条件性基因载体构建成功,在体外细胞系中,检测到rBDNF及EGFP强表达,为转基因显微注射打下基础。

Objective To construct and identify the conditional rBDNF transgenic vector in vitro. Methods rBDNF fragment by PCR amplification was subcloned into pNAO vector（ lox-Stop-lox-IRES-EGFP）, pNAO-BDNF （lox-Stop-lox-rBDNF-IRES-EGFP） and pGK-Cre were cotransfected into HEK-293 cell lines by Fugene 6. The Cre/lox recombination was confirmed by fluorescence microscopy,PCR and ELISA. Results Digested by restriction enzymes and sequenced, the targeting vector was identified. In cotransfected HEK-293 cell lines, strong （ enhanced green fluorescence protein, EGFP） fluorescence was visualized by fluorescence microscopy; Cre-lox recombination was detected by PCR in cell genomic DNA; quantitative examination of rBDNF by ELISA showed much endogenous rBDNF released into extracellular. Conclusion Conditional rBDNF transgenic vector was successfully constructed and strong overexpression of rBDNF and EGFP were identified in the cotransfected cell line, and establishment of this targeting vector is critical for pronuclear microinjection.