嘌呤霉素抗性基因捕获载体的构建及在细胞中的功能验证

目的构建具有嘌呤霉素抗性基因捕获载体，扩大基因捕获载体的应用范围。方法用经改造的捕获载体（gene trapping vector）稳定转染HepG2．2．15肝癌细胞系，经嘌呤霉素筛选，制作单克隆细胞株。用PCR方法验证该载体的在细胞染色体中的整合，ELISA方法证明捕获载体捕获基因后的细胞的功能改变。结果嘌呤霉素抗性基因捕获载体整合在HepG2．2．15肝癌细胞的染色体上，并能影响细胞HBsAg和HBeAg的分泌。结论新构建的嘌呤霉素抗性基因捕获载体能在具有G418抗性的细胞中捕获有意义的目的基因。

Construction and Functional Validation of Puromycin Resistance Gene Trapping Vector

Objective This study was aimed to construct and make use of a new gene trapping vector. Methods We reconstructed a new gene trapping vector carrying a puromycin fragment and transfected into HepG2.2. 15 cells to generate some stably transfected cells under puromycin selec- tion. PCR method was used to confirm the integration of the trapping vector in cellular chromosome. ELISA was performed to identify the functional changes in stable cell lines compared with parental HepG2.2. 15 cells. Results The newly reconstructed gene trapping vector was able to integrate in- to genomic DNA of HepG2.2. 15 cells and influence expression of HBsAg and HBeAg of HepG2.2. 15 cells. Conclusion This new reconstructed gene trapping vector can trap significant genes in G418 resistance cell lines.