靶向HIWI基因的shRNA真核表达载体的构建及鉴定

Construction and identification of shRNA eudaryotic expression vectors targeting to HIWI gene

目的构建靶向HIWI基因的shRNA真核表达载体质粒,为利用RNA干扰技术探索HIWI基因的作用的研究做准备。方法根据HIWI mRNA序列设计并合成shRNA寡核苷酸片段,退火形成双链并连接入pGenesil-2载体,并进行酶切鉴定和测序。结果酶切证明构建的shRNA已插入载体中,经测序证明与设计相同。结论成功构靶向HIWI基因的shRNA真核表达载体,为进一步研究HIWI基因在干细胞和肿瘤细胞中的作用机制及后续的体内外RNAi实验研究奠定了基础。

Objective To construct short-hairpin RNA（shRNA）eukaryotic expressionvectors targeting to HIWI gene and prepare for exploring the function of HIWI gene with RNAinterference （RNAi） technique. Methods The shRNA oligonucleotide fragments were designedand synthesized based on the sequence of HIWImRNA. Double strands were formed afterannealing and inserted intopGenesil-2 vetor. The recombinant wastransfonned into DHSa. Thenplas were extracted and identified by restriction enzyme and sequencing analysis. Results The restriction enzyme analyses demonstrate that shRNA have been inserted into vectors, and sequencing analyses demonstrate that shRNA have been inserted into vectors, and sequencing analyses demonstrate flint their sequences were the same as the design. Conclusion We have successfully constructed shRNA eukaryotic expression vectors targeting to HIWI gene. Itlaid the foundations for the further research on the function of HIWI gene in stem cell and tumorcell and the future RNAi experimental research in vivo and in vitro.