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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811
“The Nobel Prize in Physiology or Medicine 2006”. Nobelprize.org. Nobel Media AB (2013) http://www.nobelprize.org/nobel_prizes/medicine/laureates/2006/
Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864
Lee Y, Ahn C, Han J et al (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419
Hutvágner G, McLachlan J, Pasquinelli AE et al (2001) A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7small temporal RNA. Science 293:834–838
Brennecke A, Stark RB, Russell SM et al (2005) Principles of microRNA-target recognition. PLoS Biol 3:e85
Origene HuSH shRNA plasmid panels application guide OriGene Technologies (Rockville, MD)
O’Keefe EP (2013) Nucleic acid delivery: lentiviral and retroviral vectors. Labome.com
Singh RK, Lokeshwar BL (2011) The IL-8-regulated chemokine receptor CXCR7 stimulates EGFR signaling to promote prostate cancer growth. Cancer Res 71:3268–3277
Shamaladevi N, Lyn DA, Escudero DO et al (2009) CXC receptor-1 silencing inhibits androgen-independent prostate cancer. Cancer Res 69:8265–8274
Singh RK, Lokeshwar BL (2009) Depletion of intrinsic expression of Interleukin-8 in prostate cancer cells causes cell cycle arrest, spontaneous apoptosis and increases the efficacy of chemotherapeutic drugs. Mol Cancer 8:57. doi:10.1186/1476-4598-8-57
Acknowledgement
This work was funded by grants from the National Institutes of Health (1R01CA61038-14, 1R01 CA 156778–01, VA MERIT Award No 5312–01 and 5312–02, all to BLL). The authors thank Ms. Maite Lopez for illustration in Fig. 1.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Salazar, N., Muñoz, D., Hoy, J., Lokeshwar, B.L. (2014). Use of shRNA for Stable Suppression of Chemokine Receptor Expression and Function in Human Cancer Cell Lines. In: Vancurova, I. (eds) Cytokine Bioassays. Methods in Molecular Biology, vol 1172. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0928-5_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0928-5_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0927-8
Online ISBN: 978-1-4939-0928-5
eBook Packages: Springer Protocols