Gateway技术构建转基因重组质粒pRP.EX3d-EF1A-LRP16-His-IRES-eGFP

摘要

Objective To lay the foundation for establishing transgenic mice by constructing recombinant transgenic vector pRP. EX3d-EF1A-LRP16-His-IRES-eGFP. Methods The attB1- LRP16-His-attB2 was amplified by overlap PCR. The LRP16 gene was cloned into pDown vector via BP reaction and transmitted into pRP.EX3d vector via LR reaction with pDown-LRP16-His, pUp-EF1A, pTail-IRES-eGFP and PRP. Des3d. The recombinant plasmid was transformed into Stbl3 cells and screened using the ampicillin (AMP) resistance gene. Positive clones were confirmed by PCR and DNA sequencing, respectively. Results Sequencing and restriction endonuclease showed that the recombinant vector pRP.EX3d-EF1A-LRP16-His-IRES-eGFP was successfully constructed. Conclusion The successfully constructed recombinant vector pRP.EX3d-EF1A-LRP16-His-IRES-eGFP can be used in establishing LRP16 transgenic mice.%目的：构建转基因重组质粒pRP.EX3d-EF1A-LRP16-His-IRES-eGFP，为转基因鼠的建立奠定基础。方法利用重叠PCR技术扩增attB1-LRP16-His-attB2，经BP反应，将LRP16基因插入到载体pDown上，pUp-EF1A、pDown-LRP16-His、pTail-IRES-eGFP和PRP.Des3d经LR反应，将LRP16转移到pRP.EX3d上，转化Stbl3细胞，用氨苄西林进行抗性筛选，经PCR鉴定后将阳性克隆送测序。结果测序及酶切结果验证获得正确的pRP.EX3d-EF1A-LRP16-His-IRES-eGFP重组质粒。结论成功构建pRP.EX3d-EF1A-LRP16-His-IRES-eGFP重组质粒，可用于下一步LRP16转基因小鼠的构建。