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

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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

Figure 5.C0.1 Differentiation of ESCs leads to morphological changes. Examples of undifferentiated ESCs (left panel), partially differentiated ESCs (middle panel), and differentiated ESCs (right panel) are shown. This figure is adapted from Wang et al. ().

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Figure 5.C0.2 Examples of the ALP/AB ratio in a shRNA screen and target gene selection. Candidate genes identified with two independent shRNAs with Z‐scores for the ALP/AB ratio less than −1.5 or more than 1.5 may be viewed as positive or negative regulators of ESC renewal. For example, PI3K, Nme6, and Nme7, each of which have two independent shRNAs with Z‐scores that are lower than −1.5, were identified as positive regulators. This figure is adapted from Wang et al. ().

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Figure 5.C0.3 Examples of undifferentiated, partially differentiated, and fully differentiated cells scored by the morphological changes in shRNA‐targeted ESCs. This figure is adapted from Wang et al. ().

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Figure 5.C0.4 Quantification of Oct4 fluorescent signals in undifferentiated and differentiated ESCs. Indirect immunofluorescence assays were performed with anti‐Oct4 antibody in 96‐well plates and analyzed by automatic fluorescence microscopy (Cellomics). (A ) Histogram. Each dashed line represents the distribution of the intensity of Oct4 protein staining in one well. Solid line represents the average of three different wells. (B ) The quantification of fluorescence intensity.

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