title: 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.
authors.affiliation: Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, Miami, FL, USA.
authors.forename: Nicole
authors.initials: N
authors.lastname: Salazar
authors.forename: Daniel
authors.initials: D
authors.lastname: Muñoz
authors.forename: James
authors.initials: J
authors.lastname: Hoy
authors.forename: Bal L
authors.initials: BL
authors.lastname: Lokeshwar
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002999:Clone Cells; D015870:Gene Expression; D006801:Humans; D008055:Lipids / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D054387:Receptors, CXCR / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: C501896:ACKR3 protein, human; D000906:Antibodies; D008055:Lipids; C086724:Lipofectamine; D034741:RNA, Small Interfering; D054387:Receptors, CXCR