Abstract
Identifying the genes and pathways that regulate self-renewal and differentiation in somatic stem cells is a central goal in stem cell and cancer biology. Here, we describe a method for RNAi-based screens in primary human hematopoietic stem and progenitor cells. These cells are suitable targets for complex, selection-based screens using pooled lentiviral shRNA libraries. The screening approach is a promising new tool to dissect regulatory mechanisms in hematopoietic and somatic stem cells, in general, and may be particularly useful to identify gene targets and modifiers that can be further exploited in strategies for ex vivo stem cell expansion.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Berns, K., Hijmans, E. M., Mullenders, J., Brummelkamp, T. R., Velds, A., Heimerikx, M., Kerkhoven, R. M., Madiredjo, M., Nijkamp, W., Weigelt, B., Agami, R., Ge, W., Cavet, G., Linsley, P. S., Beijersbergen, R. L., and Bernards, R. (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437.
Moffat, J., Grueneberg, D. A., Yang, X., Kim, S. Y., Kloepfer, A. M., Hinkle, G., Piqani, B., Eisenhaure, T. M., Luo, B., Grenier, J. K., Carpenter, A. E., Foo, S. Y., Stewart, S. A., Stockwell, B. R., Hacohen, N., Hahn, W. C., Lander, E. S., Sabatini, D. M., and Root, D. E. (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298.
Paddison, P. J., Silva, J. M., Conklin, D. S., Schlabach, M., Li, M., Aruleba, S., Balija, V., O’Shaughnessy, A., Gnoj, L., Scobie, K., Chang, K., Westbrook, T., Cleary, M., Sachidanandam, R., McCombie, W. R., Elledge, S. J., and Hannon, G. J. (2004) A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431.
Westbrook, T. F., Martin, E. S., Schlabach, M. R., Leng, Y., Liang, A. C., Feng, B., Zhao, J. J., Roberts, T. M., Mandel, G., Hannon, G. J., Depinho, R. A., Chin, L., and Elledge, S. J. (2005) A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837–848.
Woods, N. B., Fahlman, C., Mikkola, H., Hamaguchi, I., Olsson, K., Zufferey, R., Jacobsen, S. E., Trono, D., and Karlsson, S. (2000) Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells. Blood 96, 3725–3733.
Ali, N., Karlsson, C., Aspling, M., Hu, G., Hacohen, N., Scadden, D. T., and Larsson, J. (2009) Forward RNAi screens in primary human hematopoietic stem/progenitor cells. Blood 113, 3690–3695.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Karlsson, C., Larsson, J., Baudet, A. (2010). Forward RNAi Screens in Human Stem Cells. In: Zhang, B., Stellwag, E. (eds) RNAi and microRNA-Mediated Gene Regulation in Stem Cells. Methods in Molecular Biology, vol 650. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-769-3_3
Download citation
DOI: https://doi.org/10.1007/978-1-60761-769-3_3
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-768-6
Online ISBN: 978-1-60761-769-3
eBook Packages: Springer Protocols