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RNA interference is a powerful genetic approach for efficiently silencing target genes. Expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes. This protocol describes a method to stably integrate shRNA constructs with pSUPER.retro vectors and Lipofectamine in RKO and H1299 cells. This method can be applied to cells from other lines with modification of drug selection and cell conditions. pSUPER.retro vectors can be also transfected into packaging cells by this same method to produce retroviral supernatants.
Materials and Reagents
This protocol was adapted from Cecchinelli et al. (2006) Mol Cell Biol 26:12 4746-4757. This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (to S. Soddu). I am particularly grateful to Dr. Cinzia Rinaldo and Dr. Andrea Prodosmo for technical support.
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