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.
dsRNA, Double-stranded RNA; siRNA, small interfering RNA; dsRNA, double-stranded RNA; shRNA, small hairpin RNA; egfp, enhanced green fluorescent protein gene; MEF, 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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