title: Identification of pathogenetically relevant genes in lymphomagenesis by shRNA library screens.
abstract: RNA interference (RNAi) is a conserved posttranscriptional gene silencing mechanism that has recently emerged as a breakthrough genetic tool in functional genomics and drug target discovery. An increasing number of studies applying RNAi in high-throughput screens have begun to unravel complex signaling networks underlying diverse cellular processes. This chapter describes an approach to construct a conditional small-hairpin (sh)RNA library and its application in human lymphoma cell lines. A library cloning procedure outlines the incorporation of shRNA sequences and random 60-mer "bar code" oligonucleotides, enabling rapid identification of the hairpin by microarrays. Lymphoma cell lines are optimized for efficient retroviral transduction and tetracycline inducibility. The shRNA library is suitable for identifying molecular targets in cancer, but also versatile for various screening strategies.
authors.affiliation: Division of Hematopoietic Stem Cell and Leukemia Research, Beckman Research Institute of City of Hope, Duarte, CA, USA. vngo@coh.org
authors.forename: Vu N
authors.initials: VN
authors.lastname: Ngo
mesh_terms: D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005456:Fluorescent Dyes / Q000378:metabolism; D015723:Gene Library; D005821:Genetic Techniques; D057809:HEK293 Cells; D006801:Humans; D008223:Lymphoma / Q000235:genetics* / Q000473:pathology*; D009693:Nucleic Acid Hybridization / Q000187:drug effects; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D034622:RNA Interference; D015347:RNA Probes / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D014161:Transduction, Genetic
chemical_list: D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D015347:RNA Probes; D034741:RNA, Small Interfering; D013752:Tetracycline