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

doi:10.1007/978-1-4939-0928-5_19

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 journal › Article › peer-review

10 Scopus citations

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.

Original language	English (US)
Pages (from-to)	209-218
Number of pages	10
Journal	Methods in Molecular Biology
Volume	1172
DOIs	https://doi.org/10.1007/978-1-4939-0928-5_19
State	Published - 2014
Externally published	Yes

Keywords

CXCR7
Lipofectamine 2000
Selection
ShRNA
Stable transfection

ASJC Scopus subject areas

Molecular Biology
Genetics

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1007/978-1-4939-0928-5_19

Cite this

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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 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.",

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doi = "10.1007/978-1-4939-0928-5_19",

language = "English (US)",

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T1 - Use of shrna for stable suppression of chemokine receptor expression and function in human cancer cell lines

AU - Salazar, Nicole

AU - Muñoz, Daniel

AU - Hoy, James

AU - Lokeshwar, Bal L.

N1 - Publisher Copyright: © Springer Science+Business Media New York 2014.

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - CXCR7

KW - Lipofectamine 2000

KW - Selection

KW - ShRNA

KW - Stable transfection

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JO - Methods in Molecular Biology

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

Abstract

Keywords

ASJC Scopus subject areas

UN SDGs

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