African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12198

Full Length Research Paper

Silencing effect of shRNA expression vectors with stem length of 21, 27 or 29 bp

Zhonghua Liu1, Hongwei Xiao2,3, Xinmin Zheng2,3*, Xianfeng Qiao2,3 and Huayan Wang1
  1Shaanxi Stem Cell Engineering and Technology Research Center, Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Veterinary Medicine, Northwest  A and F University, Yangling 712100, China. 2Institute of Animal and Veterinary Science, Hubei Academy of Agricultural Science, Wuhan 430064, China. 3Hubei Key Lab of Animal Embryo and Molecular Breeding, Wuhan 430064, China.
Email: [email protected]

  •  Accepted: 03 January 2011
  •  Published: 14 February 2011

Abstract

 

In this study, shRNA vectors having different stem length were constructed and their silencing effect was tested in mouse embryonic fibroblast and in vivo. Interfering RNAs were designed with stems of 21, 27, and 29 bp. The enhanced green fluorescent protein gene was used as target gene. The synthesized single strands were annealed and cloned into psiSTRIKE. Then, the recombinant plasmids were transfected into mouse embryonic fibroblast with lipofection and injected into leg muscle of mouse. The mRNA expression level of the green fluorescent protein gene was checked by real-time quantitative polymerase chain reaction (RT-PCR). The silencing effect of the 29 bp shRNA vector was stronger than that of the 21 and 27 bp in cell, but there was no significant difference among them when injected in muscle. The findings in this study are of interest for selecting the best hairpins for mouse individuals.

 

Key words: Gene silenging, shRNA, enhanced green fluorescent protein, mouse embryonic fibroblast.

Abbreviation

dsRNA, Double-stranded RNA; siRNA, small interfering RNA; dsRNA, double-stranded RNA; shRNA, small hairpin RNA; egfp, enhanced green fluorescent protein geneMEF, mouse embryonic fibroblasts; BLAST, Basic Local Alignment Search Tool; DMEM, Dulbecco’s modified Eagle’s minimal essential medium; FBS, fetal bovine serum.

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