title: Array-based high-throughput screening in mouse embryonic stem cells with shRNAs.
abstract: High-throughput short-hairpin RNA (shRNA) lentivirus screening is a powerful tool for identifying multiple functional regulators in embryonic stem cells (ESCs). shRNA libraries can efficiently down-regulate target genes persistently with high efficiency. The concurrent measurement of relative cell number by alamarBlue (AB) assay and undifferentiated ESC markers via an alkaline phosphatase (ALP) activity assay in the same cell culture well provides an efficient and economical way to pinpoint factors crucial for ESC pluripotency and/or expansion. Most of the renewal pathways affect ALP activity. Thus, multiple positive and negative regulators can be identified by this method. In addition, morphological changes and/or the expression levels of specific pluripotency or differentiation markers examined by immunofluorescence can be used as secondary screens for target-gene selection. In summary, we describe an efficient way to identify multiple regulators of ESC renewal using shRNAs. Curr. Protoc.
authors: Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors: Chia-Hui
authors: CH
authors: Wang
authors: Institute of Systems Biology and Bioinformatics, National Central University, Taoyuan, Taiwan.
authors: Nianhan
authors: N
authors: Ma
authors: Genomics Research Center, Academia Sinica, Taipei, Taiwan.|Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
authors: Yu-Tsen
authors: YT
authors: Lin
authors: Genomics Research Center, Academia Sinica, Taipei, Taiwan.|Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
authors: Cheng-Chung
authors: CC
authors: Wu
authors: Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors: Hong-Jin
authors: HJ
authors: Wu
authors: Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors: Ching-Chia
authors: CC
authors: Yu
authors: Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors: Michael
authors: M
authors: Hsiao
authors: Department of Pediatrics, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan.
authors: Frank Leigh
authors: FL
authors: Lu
authors: Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan County, Taiwan.
authors: Scott C
authors: SC
authors: Schuyler
authors: Genomics Research Center, Academia Sinica, Taipei, Taiwan.|Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.|Genomics and System Biology Program, College of Life Science, National Taiwan University, Taipei, Taiwan.|National Core Facility Program for Biotechnology, National RNAi Platform, Taipei, Taiwan.
authors: Jean
authors: J
authors: Lu
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D048430:Cell Shape; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D057075:Enzyme Assays; D061252:Feeder Cells / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D057166:High-Throughput Screening Assays / Q000379:methods*; D016086:Lentivirus / Q000378:metabolism; D051379:Mice; D046228:Microarray Analysis / Q000379:methods*; D010078:Oxazines / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection; D014966:Xanthenes / Q000378:metabolism
chemical_list: D010078:Oxazines; D034741:RNA, Small Interfering; D014966:Xanthenes; C005843:resazurin; D000469:Alkaline Phosphatase
keywords: RNA interference; embryonic stem cells; high-throughput screening; short-hairpin RNA