pLKO.1-ZNF703 shRNA质粒的构建与鉴定

Construction and identification of pLKO.1-ZNF703 shRNA plasmid

关键词:  ZNF703  pLKO.1 质粒  shRNA  HCT-116 细胞

Key words:  ZNF703  pLKO.1 plasmid  shRNA  HCT-116 cells

摘要:

摘　要：目的　构建pLKO.1-ZNF703 shRNA质粒靶向敲低ZNF703。方法　根据人和鼠ZNF703 基因序列中的外显 子区在RNA干扰平台上（broadinstitute.org）设计shRNA的靶向序列，并根据pLKO.1 中 Age I 和EcoR I酶切位点序列设 计并合成ZNF703 shRNA的引物；对引物进行退火；将pLKO.1 质粒进行酶切、胶回收；用T4 酶进行连接、转化、涂板，对 阳性克隆菌落PCR鉴定和测序鉴定；将构建好的质粒利用慢病毒包装并转染HCT-116 细胞系，Q-PCR检测ZNF703 的 敲低效率。结果　成功构建pLKO.1-ZNF703 shRNA质粒，并且利用病毒包装体系构建ZNF703 shRNAHCT-116 细胞系， RT-PCR结果显示设计并构建的pLKO.1-ZNF703 shRNA 质粒可用于有效敲低ZNF703。结论　成功构建可用于有效敲低 人或鼠源细胞系中ZNF703 的pLKO.1 -ZNF703 shRNA质粒。

Abstract:

Abstract: Objective To construct pLKO.1-ZNF703 shRNA plasmid for effectively knockdown ZNF703. Methods The targeting sequences of shRNA were designed on the RNA interference platform (broadinstitute.org) based on the exon region of human and mouse ZNF703 gene sequences in human and mice. The primers of ZNF703 shRNA were designed and synthesized according to the sequences of Age I and EcoR I enzyme cleavage sites in pLKO.1. The primes were then annealed. The pLKO.1 plasmid underwent enzyme digestion and gel purification. T4 DNA ligase was used for ligation, transformation and plate coating, and the positive colonies underwent PCR identification and sequencing analysis. The plasmid successfully constructed was packed with the lenti-virus system and transfected to HCT-116 cell lines. The Q-PCR was used to detect the knockdown efficiency of ZNF703. Results The pLKO.1-ZNF703 shRNA plasmid was successfully constructed, and the ZNF703 shRNA HCT-116 cell lines was constructed with virus packaging system. The RT-PCR results showed that the pLKO.1-ZNF703 shRNA plasmid designed and constructed can be used to effectively knock down ZNF703. Conclusion The pLKO.1-ZNF703 shRNA plasmid that can be used to effectively knock down ZNF703 in human and mouse cell lines has been successfully constructed.