Abstract
Here, we describe the use of a functional cloning approach, based on the screen of a genomewide short hairpin RNA (shRNA) library, to identify novel genes regulating apoptosis in neuronal cells. Apoptosis is induced by doxorubicin and is detected with a fluorometric caspase 3 assay. Moreover, we describe also the screen of the library to identify genes regulating the processing of the β-amyloid (Aβ) precursor protein (APP), the protein associated with the pathogenesis of Alzheimer’s disease. The levels of the peptide Aβ, produced by the APP processing, are detected with ELISA based on the innovative Delfia method developed by PerkinElmer.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hannon, G.J. (2002) RNA interference. Nature 418, 244–251.
Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D.P., Zipperlen, P., and Ahringer, J. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237.
Kiger, A.A., Baum, B., Jones, S., Jones, M.R., Coulson, A., Echeverri, C., and Perrimon, N. (2003) A functional genomic analysis of cell morphology using RNA interference. J Biol 2, 27.
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.
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.
Mattson, M.P. (2004) Pathways towards and away from Alzheimer’s disease. Nature 430, 631–639. Addendum in (2004) Nature 431, 107.
Author information
Authors and Affiliations
Editor information
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Visconti, R., D’Adamio, L. (2007). Functional Cloning of Genes Regulating Apoptosis in Neuronal Cells. In: Borsello, T. (eds) Neuroprotection Methods and Protocols. Methods in Molecular Biology, vol 399. Humana Press. https://doi.org/10.1007/978-1-59745-504-6_9
Download citation
DOI: https://doi.org/10.1007/978-1-59745-504-6_9
Publisher Name: Humana Press
Print ISBN: 978-1-58829-666-5
Online ISBN: 978-1-59745-504-6
eBook Packages: Springer Protocols