不同长度茎部shRNA表达载体构建与鉴定

Construction and identification of different stem shRNA expression vectors

摘要:

本研究针对同一目的基因设计构建不同茎部长度的shRNA表达载体，并对其在细胞及胚胎水平的干扰效应做一比较。以绿色荧光蛋白基因为沉默效应的靶基因，设计茎部长度分别为21 bp、27 bp、29 bp的干扰片段，退火后连入带有H6启动子的真核表达载体psiSTRIKE中 (分别命名为EGFP-21 siRNA、EGFP-27 siRNA和EGFP-29 siRNA)，将构建成功的载体以脂质体法转染小鼠胚胎成纤维细胞，利用荧光定量PCR对其荧光表达进行精确定量。不同茎部长度的shRNA载体均使绿色荧光蛋白基因表达降低，茎部为29 bp时比21 bp、27 bp表现出更明显的沉默效应。细胞水平沉默效应的初步验证，为筛选适合小鼠个体水平的最佳发夹结构奠定了基础。

Abstract:

We constructed shRNA vectors with different stem length, and tested the silencing effectiveness in mouse cells and embryos. We designed interfering RNAs with stems of 21 bp, 27 bp, and 29 bp. The enhanced green fluorescent protein gene was used as target gene. The synthesized single strands were annealed and cloned into psiSTRIKE and the recombinant plasmids (EGFP-21 siRNA, EGFP-27 siRNA, and EGFP-29 siRNA) were transfected into the mouse embryonic fibroblast with lipofection. The mRNA expression level of the enhanced green fluorescent protein gene was checked by real-time quantitative PCR. The silencing effectiveness of the 29 bp shRNA vector was stronger than which of the 21 bp and 27 bp. The findings in this study are of interest for selecting the hairpins for mouse individuals.