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Gene silencing in mammalian cells by PCR‐based short hairpin RNA
Author(s) -
Gou Deming,
Jin Nili,
Liu Lin
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)00630-6
Subject(s) - rna interference , biology , small interfering rna , microbiology and biotechnology , small hairpin rna , gene silencing , rna silencing , gene , rna , subcloning , transfection , genetics , plasmid
RNA interference (RNAi) provides a powerful tool to silence genes in a sequence‐specific manner in a variety of systems. However, not all sequences are effective in the RNAi‐mediated gene silencing. In this study, we developed a polymerase chain reaction (PCR)‐based RNAi strategy for a quick screening of small interfering RNA (siRNA) efficiency. This method utilized a two‐step PCR to generate a chimeric DNA template containing the U6 promoter or cytomegalovirus promoter and short hairpin DNA. We demonstrated that the transfection of the PCR products into mammalian cells resulted in specific depressions of exogenous (luciferase, green fluorescent protein and β‐galactosidase) and endogenous (annexin II) gene expressions. This PCR strategy provides a rapid, easy and cheap approach for testing candidates siRNA sequences and is an attractive alternative to subcloning.