A miR-21 hairpin structure-based gene knockdown vector.
RNA interference (RNAi) is widely used to study gene functions as a reverse genetic means from first-generation siRNA to second-generation short hairpin RNA (shRNA) or the newly developed microRNA (shRNA-miR). Here we report a gene knockdown vector system based on the mouse miR-21 hairpin structure. In this system, the pre-miRNA hairpin of the miR-21 gene was modified by replacing the 22-nucleotide mature sequence with shRNA sequences that target genes of interest, flanked by 160-bp upstream and 65-bp downstream sequences of the mouse pre-miR-21. We tested this system by knocking down the enhanced green fluorescence protein (EGFP) reporter gene using different vectors, in which shRNA-miR was driven by the polymerase II (pol II) promoter. We found that miR-21 hairpin-based shRNA-miR can be directly placed under pol II promoter, like UbC or CMV promoter to knockdown the gene of interest. To facilitate the wide application of the miR-21 hairpin-based gene knockdown system, we further knocked down the endogenous gene lamin (A/C), which showed that endogenous lamin A/C expression can be efficiently silenced using the miR-21 hairpin-based lentiviral vector. The miR-21 hairpin-based gene knockdown vector will provide a new genetic tool for gene functional studies in vitro and in vivo.
Yue
Junming
J
Department of Physiology, University of Tennessee Health Science Center, 19 S. Manassas St., Memphis, TN 38163, USA. jyue@uthsc.edu
Sheng
Yi
Y
Ren
Aixia
A
Penmatsa
Sravya
S
D000818:Animals; D001483:Base Sequence; D004257:DNA Polymerase II / Q000235:genetics; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D034904:Lamin Type A / Q000235:genetics; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic
D034904:Lamin Type A; C531950:MIRN21 microRNA, mouse; D035683:MicroRNAs; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004257:DNA Polymerase II