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

Nicole Salazar, Daniel Muñoz, James Hoy, Bal L. Lokeshwar

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)209-218
Number of pages10
JournalMethods in molecular biology (Clifton, N.J.)
Volume1172
DOIs
StatePublished - 2014
Externally publishedYes

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Chemokine Receptors
Small Interfering RNA
Cell Line
Neoplasms
Puromycin
Immunoblotting
Transfection
Plasmids
Anti-Bacterial Agents
Rabbits
Lipids
Polymerase Chain Reaction
Messenger RNA
Proteins

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Use of shRNA for stable suppression of chemokine receptor expression and function in human cancer cell lines. / Salazar, Nicole; Muñoz, Daniel; Hoy, James; Lokeshwar, Bal L.

In: Methods in molecular biology (Clifton, N.J.), Vol. 1172, 2014, p. 209-218.

Research output: Contribution to journalArticle

Salazar, Nicole ; Muñoz, Daniel ; Hoy, James ; Lokeshwar, Bal L. / Use of shRNA for stable suppression of chemokine receptor expression and function in human cancer cell lines. In: Methods in molecular biology (Clifton, N.J.). 2014 ; Vol. 1172. pp. 209-218.
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