Use of shrna for stable suppression of chemokine receptor expression and function in human cancer cell lines

Abstract

In this chapter, we describe a protocol used for stable silencing of chemokine receptor CXCR7 in humancancer cells using shRNA in a lipid transfection setting, previously published by our laboratory. We providethorough detail and background information about the process of shRNA to clarify the importance of thisprocess. We use CXCR7 shRNA and scrambled sequence shRNA constructs cloned into a pRS plasmidunder the control of a U6 promoter for stable expression. Human cancer cells are transfected with shRNApRSusing Lipofectamine 2000. Cells stably expressing the shRNA are selected from transfected culturesfollowing 2 weeks in medium containing the selection antibiotic puromycin. The emergent cell coloniesare evaluated for knockdown of CXCR7 mRNA and protein expression by q-PCR and immunoblottingwith rabbit anti-CXCR7 IgG, respectively.