Array‐Based High‐Throughput Screening in Mouse Embryonic Stem Cells with shRNAs

Chia‐Hui Wang1, Nianhan Ma2, Yu‐Tsen Lin3, Cheng‐Chung Wu3, Hong‐Jin Wu1, Ching‐Chia Yu1, Michael Hsiao1, Frank Leigh Lu4, Scott C. Schuyler5, Jean Lu6

1 Academia Sinica, Taipei, 2 National Central University, Taoyuan, 3 National Defense Medical Center, Taipei, 4 National Taiwan University Hospital and National Taiwan University Medical College, Taipei, 5 Chang Gung University, Taoyuan County, 6 National RNAi Platform, Taipei
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5C.3
DOI:  10.1002/9780470151808.sc05c03s26
Online Posting Date:  September, 2013
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Abstract

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. Stem Cell Biol. 26:5C.3.1‐5C.3.19. © 2013 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; RNA interference; short‐hairpin RNA; high‐throughput screening

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Infection of Feeder‐Free Mouse ESCs with an shRNA Lentivirus Library
  • Basic Protocol 2: Select the Candidate Genes by alamarBlue Assay and Alkaline Phosphatase Assay
  • Basic Protocol 3: Select the Candidate Genes Based on Morphological Changes by shRNA Infection
  • Alternate Protocol 1: Select the Candidate Genes Based on Morphological Changes by Transfection
  • Alternate Protocol 2: Selecting Candidate Genes by Immunofluorescence Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Infection of Feeder‐Free Mouse ESCs with an shRNA Lentivirus Library

  Materials
  • Mouse ESCs (unit 1.4)
  • Mouse ESC medium (for ESC cultured on feeders; see receipe)
  • Feeder‐free mouse ESC culture medium (see recipe)
  • STO cells (ATCC, cat. no. CRL‐1503)
  • STO medium (see recipe)
  • 10 µg/ml mitomycin C (Sigma, cat. no. M4287) in STO medium
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.05% trypsin‐EDTA (Invitrogen, cat. no. 25200‐056) prepared in PBS
  • Protamine sulfate (Sigma, cat. no. P4505)
  • Appropriate selection antibiotic (see documentation supplied with library or vector)
  • shRNA library (can be purchased from Sigma or obtained through the cooperation project with the RNAi Consortium, or the core facility at an institution; the lentivirus production and titration protocol can be downloaded from: http://www.broadinstitute.org/rnai/public/resources/protocols)
  • 100‐mm tissue culture dishes (Corning, cat. no. 430167), gelatin coated (see recipe for gelatin coating)
  • Automatic cell counter and slides, or trypan blue solution and hemacytometer
  • 96‐well plates (Corning, cat. no. 3599)
  • Multidrop Combi Reagent Dispenser (Thermo, cat. no. 5840300): before use, sterilize the tubing of the Multidrop with 100 ml of 70% ethanol, 100 ml sterile water, and 100 ml sterile PBS, sequentially—after seeding cells, wash the tube with 100 ml sterile water, then 100 ml 70% ethanol (it is also possible to wash and autoclave the tube before or after use)
  • Multichannel pipettor (Thermo, cat. no. 46300300; optional alternative to Multidrop and liquid handling system) and sterile reagent reservoir
  • Liquid handling system (Beckman Coulter, cat no. Biomek NX or Tecan, cat. no. freedom EVO) in a biological safety cabinet
  • Additional reagents and equipment for culturing mouse ESC (unit 1.4)

Basic Protocol 2: Select the Candidate Genes by alamarBlue Assay and Alkaline Phosphatase Assay

  Materials
  • shRNA lentivirus‐infected ESCs (see protocol 1)
  • Feeder‐free ESC culture medium (see recipe, optional)
  • alamarBlue solution (Biotium cat. no. 30025, or AbD Serotec, BUF012B)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 100% methanol
  • p‐nitrophenyl phosphate liquid substrate (Sigma; aliquot and store at −20°C)
  • Inverted microscope equipped with 4×, 10×, and 20× lenses
  • Multichannel pipettor (Thermo, cat. no. 46300300; optional alternative to Multidrop and liquid handling system) and sterile reagent reservoir
  • Liquid handling system (Beckman Coulter, cat no. Biomek NX or Tecan, cat. no. freedom EVO) in a biological safety cabinet
  • Spectrophotometer or fluorometer (excitation wavelength, 530 to 560 nm; emission wavelength, 590 nmWallac Victor3, PerkinElmer:)
  • Microsoft Excel program

Basic Protocol 3: Select the Candidate Genes Based on Morphological Changes by shRNA Infection

  Materials
  • shRNA lentivirus‐infected ESCs (see protocol 1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Feeder‐free ESC culture medium (see recipe)
  • Inverted microscope equipped with 4×, 10×, and 20× lenses
  • Liquid handling system (Beckman Coulter, cat no. Biomek NX, or Tecan, cat. no. freedom EVO) or multichannel pipettor (Thermo, cat. no. 46300300) and sterile reagent reservoir
  • Microsoft Excel program
  • Additional reagents and equipment for infection of mouse ESCs with shRNA lentivirus library ( protocol 1)

Alternate Protocol 1: Select the Candidate Genes Based on Morphological Changes by Transfection

  Materials
  • Mouse ESCs cultured in feeder‐free conditions (unit 1.4)
  • Feeder‐free ESC culture medium (see recipe)
  • shRNA plasmids of the candidate genes (contain selection marker)
  • Opti‐MEM medium (Invitrogen, cat. no. 31985)
  • Lipofectamine 2000 (Invitrogen, cat. no. 11668)
  • Gelatin‐coated 6‐well or 96‐well plates
  • 96 well, V‐bottom microtiter plates (optional; Nunc, cat. no. 249946)
  • Inverted microscope equipped with 4×, 10×, and 20× lenses

Alternate Protocol 2: Selecting Candidate Genes by Immunofluorescence Assay

  Materials
  • shRNA lentivirus‐infected or plasmid‐transfected ESCs (see protocol 1, or protocol 4) seeded on 96‐well plate with clear bottom and black wall
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4% formaldehyde‐PBS (see recipe) or methanol (optional)
  • 0.1% (v/v) Triton X‐100 in PBS
  • Primary antibody
  • Bovine serum albumin or serum corresponding to the species from which the primary antibody was obtained
  • Fluorescein‐conjugated secondary antibody (Invitrogen Alexa Fluor anti‐mouse/rabbit/anti‐goat IgG)
  • Liquid handling system (Beckman Coulter, cat no. Biomek NX, or Tecan, freedom EVO) or multichannel pipettor (Thermo, cat. no. 46300300) and reagent reservoir
  • Automatic fluorescence microscopy reader (Cellomics HCS Reader, Thermo, ArrayScan VTI, or BD Biosciences, BD Pathway 435 imaging system) or fluorometer reader (Victor III, PerkinElmer)
  • Microsoft Excel program
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Figures

Videos

Literature Cited

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