Cell Biological Analysis of DT40 Knockout Cell Lines for Cell‐Cycle Genes

Aussie Suzuki1, Tatsuo Fukagawa1

1 National Institute of Genetics and The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 8.7
DOI:  10.1002/0471143030.cb0807s50
Online Posting Date:  March, 2011
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Abstract

DT40 is a chicken B cell line that has been widely used as a model for gene functional studies because the high level of homologous recombination in DT40 cells allows targeted disruption of a gene of interest. While our laboratory uses DT40 cells to understand kinetochore assembly and function, the approach is applicable to functional studies of other genes that are required for cell cycle progression. Protocols are presented for the creation of knockout cells and subsequent cell biological analyses for characterizing the phenotypes of these cells. Curr. Protoc. Cell Biol. 50:8.7.1-8.7.17. © 2011 by John Wiley & Sons, Inc.

Keywords: DT40; transfection; gene targeting; cell biological analysis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Creation of the Knockout Vector and Cell Line
  • Basic Protocol 2: Analysis of Cell Cycle Profile by Fluorescence-Activated Cell Sorting (FACS)
  • Basic Protocol 3: Indirect Immunofluorescence Analysis in DT40 Cells
  • Basic Protocol 4: Indirect Immunofluorescence Analysis of Metaphase Chromosome Spreads
  • Basic Protocol 5: Immuno-Electron Microscopy (EM) Analysis in DT40 Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Creation of the Knockout Vector and Cell Line

 Materials
  • 30 to 40 µg of knockout construct plasmid for the gene of interest/transfection, e.g., CENP-L construct (Fig. 8.7.2)
  • Restriction enzymes (e.g., NotI, SalI, and NcoI for the CENP-L construct in Fig. 8.7.2)
  • 0.7% (w/v) agarose gels
  • 1:1 (v/v) phenol/chloroform
  • 100% and 70% (v/v) ethanol
  • Phosphate-buffered saline (PBS, appendix 2A)
  • >106 DT40 cells (ATCC)/ml in suspension: culture from frozen stock in 30 ml Dulbecco's modified medium (DMEM; Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS; see appendix 2A), 1% (v/v) chicken serum, 1% (v/v) penicillin/streptomycin (P/S, Gibco) in a 75-cm2 culture flask for 3 to 4 days
  • DT40 medium: DMEM supplemented with 10% (v/v) FBS, 1% (v/v) chicken serum
  • 2× antibiotic selection medium: e.g., DT40 medium containing 1 µg/ml puromycin for knockout of CENP-L
  • 1:1 (v:v) phenol/chloroform
  • Lysis buffer without sodium dodecyl sulfate (SDS) and proteinase K: 100 mM Tris (pH 8.0; see appendix 2A)/200 mM NaCl/5 mM EDTA (see appendix 2A)
  • Lysis buffer with SDS and proteinase K: 100 mM Tris (pH 8.0)/200 mM NaCl/5 mM EDTA/0.6% (w/v) SDS/0.6 mg/ml proteinase K
  • Isopropanol
  • Tris/EDTA buffer, pH8 (TE; appendix 2A)
  • Repressible promoter system (e.g., Tet-Off, Clontech) with cDNA for the gene of interest
  • 1.5-ml microcentrifuge tubes
  • 50-ml polypropylene tubes (e.g., Corning)
  • 4-mm electropolation cuvettes (Bio-Rad)
  • Electropolator (e.g., Gene Pulser, Bio-Rad)
  • 96-well flat-bottom microtiter plates (e.g., Corning cat. no. 3596)
  • Multichannel pipettor and sterile troughs
  • 24-well flat-bottom microtiter plates (e.g., Corning cat. no. 3526)
  • 25-cm2 tissue culture flasks
  • Additional reagents and equipment for performing agarose gel electrophoresis (Voytas, 2000) and Southern hybridization (Brown, 2004), and for determining cell numbers (e.g., see unit 1.1)

Basic Protocol 2: Analysis of Cell Cycle Profile by Fluorescence-Activated Cell Sorting (FACS)

 Materials
  • 10 mM bromodeoxyuridine (BrdU)
  • 5–10 × 106 knockout (Basic Protocol 1) and control DT40 cells in 10 ml of DT40 medium in 25-cm2 tissue culture flasks
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 70% (v/v) ethanol, ice-cold
  • 1% (v/v) bovine serum albumin (BSA)/PBS (appendix 2A)
  • 2 M HCl/0.5% (v:v) Triton X-100
  • Anti-BrdU antibody (BD Biosciences, cat. no. 347580)
  • Anti-mouse IgG, FITC conjugated (Jackson Laboratory, cat. no. 115-095-003)
  • Propidium iodide (PI)
  • 15-ml polypropylene tubes (Falcon or Corning)
  • Polystyrene FACS tubes (Becton-Dickinson)
  • Flow cytometer (e.g., FACS scan, Becton-Dickinson)

Basic Protocol 3: Indirect Immunofluorescence Analysis in DT40 Cells

 Materials
  • 5–10 × 106 knockout (Basic Protocol 1) and control DT40 cells in 10 ml of DT40 medium in 25-cm2 tissue culture flasks
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 3% (w/v) paraformaldehyde (PFA)/250 mM HEPES (pH7.4)
  • 0.5% w/v NP-40 (Sigma)/PBS
  • Primary antibodies for proteins of interest (e.g., anti-CENP-T; generated in-house)
  • 0.5% (v/v) bovine serum albumin (BSA)/PBS
  • Appropriate secondary dye-conjugated antibody (e.g., FITC- or Cy3-conjugated anti-rabbit IgG, Jackson Laboratory, cat. no. 111-097-003 or 111-167-003)
  • 0.2 µg/ml 4,6-diamidino-2-phenylindole (DAPI)/Vectashield Antifade (Vector Laboratories)
  • Glass microscope slides
  • Cytospin centrifuge (e.g., Cytospin 3, Shandon)
  • Coplin jars
  • Parafilm
  • Fluorescence light microscope (e.g., Olympus IX71) with CCD camera
  • Additional reagents and equipment for determining cell numbers (e.g., see unit 1.1)

Basic Protocol 4: Indirect Immunofluorescence Analysis of Metaphase Chromosome Spreads

 Materials
  • 5–10 × 106 knockout (Basic Protocol 1) and control DT40 cells in 10 ml of DT40 medium in 25-cm2 tissue culture flasks
  • Nocodazole (e.g., Sigma, cat. no. M1404)
  • DT40 medium: Dulbecco's modified medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS; see appendix 2A)/ 1% (v/v) chicken serum
  • 0.56% hypotonic buffer (40 mM KCl/0.5 mM EDTA/20 mM HEPES, pH 7.5)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Primary antibodies for proteins of interest (e.g., anti-CENP-T; generated in-house)
  • 0.5% bovine serum albumin (BSA)/PBS (appendix 2A)
  • Appropriate secondary dye–conjugated antibody (e.g., FITC- or Cy3-conjugated anti-rabbit IgG, Jackson Laboratory, cat. no. 111-097-003 or 111-167-003)
  • 3% paraformaldehyde/250 mM HEPES (pH7.4)
  • 0.2 µg/ml of 4,6-diamidino-2-phenylindole (DAPI)/Vectashield Antifade (Vector Laboratories)
  • 15-ml polypropylene tubes (Falcon or Corning)
  • Glass microscope slides
  • Cytospin centrifuge (e.g., Cytospin 3, Shandon)
  • Coplin jars
  • Parafilm
  • Fluorescence light microscope (e.g., Olympus IX71) with CCD camera

Basic Protocol 5: Immuno-Electron Microscopy (EM) Analysis in DT40 Cells

 Materials
  • 5–10 × 106 knockout (Basic Protocol 1) and control DT40 cells in 10 ml of DT40 medium in 25-cm2 tissue culture flasks
  • Phosphate-buffered saline (PBS, appendix 2A)
  • 3% (w/v) paraformaldehyde (PFA)/250 mM HEPES, pH7.4
  • 0.5%-NP-40 (Sigma)/PBS
  • Antibodies to proteins of interest (e.g., Santa Cruz, Abcam, or MBL)
  • 0.5% (v/v) bovine serum albumin (BSA)/PBS
  • Appropriate secondary nanogold-conjugated antibody (e.g., Nanogold conjugated anti-rabbit IgG, Nanoprobes)
  • 2% (v/v) glutaraldehyde (e.g., Sigma cat. no. G7776)/100 mM sodium cacodylate buffer, pH7.3 (e.g., TAAB Laboratories)
  • 7% (w/v) sucrose (e.g., Wako)/100 mM sodium cacodylate buffer, pH7.3
  • Silver enhancer kit (e.g., HQ silver, Nanoprobes)
  • 0.5% (w/v) osmium tetroxide (OsO4; e.g., PGM Chemical)
  • Uranyl acetate (UA, e.g., Mallinckrodt)
  • 20%, 30%, 40%, 60%, 80%, 95%, and 100% ethanol
  • 50% (v/v) ethanol/propylene oxide (e.g., Wako)
  • 100% propylene oxide
  • 50% (v/v) epoxy resin (EPON; e.g., EPON 812, MNA, DDSA, DMP-30; TAAB Laboratories)/propylene oxide
  • 100% epoxy resin
  • Glass microscope slides
  • Parafilm
  • Cytospin centrifuge (e.g., Cytospin 3, Shandon)
  • Coplin jars
  • Ultramicrotome (e.g., EM UC6+FC6, Leica)
  • Grid with formvar membrane (e.g., Veco)
  • Electron microscope (e.g., JEM1010, JEOL)
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Figures

  •  FigureFigure 8.7.1 Scheme for gene disruption using homologous recombination. The knockout construct with a drug resistance gene and the homologous regions of the genome bracketing the gene of interest (indicated by the darker lines) is introduced into cells. Homologous recombination occurs, and the target gene is replaced with the knockout construct.
    View Image
  •  FigureFigure 8.7.2 Example of gene disruption. (A) Restriction maps of the CENP-L locus, the gene knockout construct, with a drug resistance gene (puror, in this example), and targeted loci. Black boxes indicate the positions of the four exons to be disrupted. SalI and NcoI restriction sites are shown, and the position of the probe used for Southern hybridization (which should be >0.5 kb) is indicated by the dark line. The novel 7.6-kb fragment digested with both SalI and NcoI hybridizes to the probe if targeted integration of the construct has occurred. (B) Restriction analysis of the targeted integration of the CENP-L disruption construct. Genomic DNA from wild-type DT40 cells and a clone after first-round targeting are analyzed by Southern hybridization with the probe indicated in (A). The wild-type allele is detected at 9.8 kb, and the target allele is detected at 7.6 kb. The molecular size markers are a HindIII digest of DNA.
    View Image
  •  FigureFigure 8.7.3 Strategy for creation of a conditional DT40 knockout cell line. The 1st-round target construct containing the puromycin resistance gene puror is transfected into DT40 cells. After 1st-round targeting, one clone is cotransfected with a cDNA construct under the control of a tet-repressible promoter and a tet-repressible transactivator (Tet-Off system) to provide conditional gene activity when the 2nd essential target allele is disrupted. Then the clone with the DNA transgene is transfected with the 2nd-round target construct containing an appropriate drug resistance gene (in this case histindinol resistance, hisD) to disrupt the remaining allele. Conditional knockout cells die after addition of tetracycline.
    View Image
  •  FigureFigure 8.7.4 Cell cycle profile of wild-type DT40 cells and CENP-T knockout (KO) cells. Cells are stained with FITC-anti-BrdU (y axis, log scale) to detect BrdU incorporation (DNA replication) and with propidium iodide to detect total DNA (x axis, linear scale). The lower-left box represents G1-phase cells, the upper box represents S-phase cells, and the lower-right box represents G2/M-phase cells. The numbers given in the boxes indicate the percentage of gated events. Experimental data are derived from Hori et al. (2008).
    View Image
  •  FigureFigure 8.7.5 Localization of CENP-T at progressive stages of the cell cycle in DT40 cells. Cells are fixed and stained with anti-CENP-T antibody (red). Nuclei and chromosomes are visualized by counterstaining with DAPI (blue). The right column shows the merged images for the DAPI and antibody-stained cells. The scale bar corresponds to 10 µm.
    View Image
  •  FigureFigure 8.7.6 Localization of CENP-T on mitotic chromosomes CENT-P signals generated by FITC-conjugated secondary antibodies are shown in red. Chromosomes are visualized by counterstaining with DAPI (blue). The scale bars correspond to 10 µm.
    View Image
  •  FigureFigure 8.7.7 Immuno-electron microscopy (EM) image of mitotic chromosomes stained with anti-CENP-E antibodies. (A) The dark signals are the result of gold labeling. CENP-E localizes to the kinetochore plate (B) Magnified image of the region in the yellow box in panel A. The scale bars correspond.
    View Image

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Literature Cited

Literature Cited
    Amano, M., Suzuki, A., Hori, T., Backer, C., Okawa, K., Cheeseman, I.M., and Fukagawa, T. 2009. The CENP-S complex is essential for the stable assembly of outer kinetochore structure. J. Cell Biol. 186:173-182.
    Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2011. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
    Brown, T. 2004. Analysis of DNA sequences by blotting and hybridization. Curr. Protoc. Mol. Biol. 68:2.9.1-2.9.20.
    Buerstedde, J.M. and Takeda, S. 1991. Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67:179-188.
    Fukagawa, T., Mikami, Y., Nishihashi, A., Regnier, V., Haraguchi, T., Hiraoka, Y., Sugata, N., Todokoro, K., Brown, W., and Ikemura, T. 2001. CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells. EMBO J. 20:4603-4617.
    Fukagawa, T. 2008. The kinetochore and spindle checkpoint in vertebrate cells. Front. Biosci. 13:2705-2713.
    Hori, T., Amano, M., Suzuki, A., Backer, C.B., Welburn, J.P., Dong, Y., McEwen, B.F., Shang, W.H., Suzuki, E., Okawa, K., Cheeseman, I.M., and Fukagawa, T. 2008. CCAN makes multiple contacts with centromeric DNA to provide distinct pathways to the outer kinetochore. Cell 135:1039-1052.
    Nishihashi, A., Haraguchi, T., Hiraoka, Y., Ikemura, T., Regnier, V., Dodson, H., Earnshaw, W.C., and Fukagawa, T. 2002. CENP-I is essential for centromere function in vertebrate cells. Dev. Cell 2:463-476.
    Jackson, A.L., Bartz, S.R., Schelter, J., Kobayashi, S.V., Burchard, J., Mao, M., Li, B., Cavet, G., and Linsley, P.S. 2003. Expression profiling reveals off-target gene regulation by RNAi. Nature Biotechnol. 21:635-637.
    Okada, M., Cheeseman, I.M., Hori, T., Okawa, K., McLeod, I.X., Yates, J.R. III, Desai, A., and Fukagawa, T. 2006. The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nature Cell Biol. 8:446-457.
    Voytas, D. 2000. Resolution and recovery of large DNA fragments. Curr. Protoc. Mol. Biol. 51:2.5A.1-2.5A.9.
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