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 human cancer cells using shRNA in a lipid transfection setting, previously published by our laboratory. We provide thorough detail and background information about the process of shRNA to clarify the importance of this process. We use CXCR7 shRNA and scrambled sequence shRNA constructs cloned into a pRS plasmid under the control of a U6 promoter for stable expression. Human cancer cells are transfected with shRNA-pRS using Lipofectamine 2000. Cells stably expressing the shRNA are selected from transfected cultures following 2 weeks in medium containing the selection antibiotic puromycin. The emergent cell colonies are evaluated for knockdown of CXCR7 mRNA and protein expression by q-PCR and immunoblotting with rabbit anti-CXCR7 IgG, respectively.