Functional cloning of genes regulating apoptosis in neuronal cells.
Here, we describe the use of a functional cloning approach, based on the screen of a genome-wide 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 f the beta-amyloid (Abeta) precursor protein (APP), the protein associated with the pathogenesis of lzheimer's disease. The levels of the peptide Abeta, produced by the APP processing, are detected with ELISA based on the innovative Delfia method developed by PerkinElmer.
Visconti
Roberta
R
Ceinge Biotecnologie Avanzate S.C.ar.l., Napoli, Italy.
D'Adamio
Luciano
L
D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000032:analysis / Q000378:metabolism; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D053148:Caspase 3 / Q000032:analysis / Q000378:metabolism; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D004317:Doxorubicin / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D051379:Mice; D009474:Neurons / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
D016564:Amyloid beta-Protein Precursor; D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; D034741:RNA, Small Interfering; D004317:Doxorubicin; C505409:Casp3 protein, mouse; D053148:Caspase 3