Human Somatic Cell Gene Targeting

Todd Waldman1, Carolyn Lee1, Tagvor G. Nishanian1, Jung‐Sik Kim1

1 Georgetown University School of Medicine, Washington, D.C.
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 9.15
DOI:  10.1002/0471142727.mb0915s62
Online Posting Date:  May, 2003
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Abstract

Human somatic cell gene targeting provides a powerful tool to scientists studying gene function in cultured human cells. This technology allows scientists to knock out genes in human somatic cells in a fashion analogous to the creation of knockout mice. Human somatic cell gene targeting brings the power of genetics to the study of human genes in human cells by making it possible to compare cells or individuals that are genetically identical except for a single, well‚Äźdefined mutation in an endogenous gene. These modified cells can be studied both in vitro and in vivo. This unit presents protocols for human somatic cell gene targeting.

     
 
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Table of Contents

  • SECTION IV: Inactivation of Genes in Mammalian Cells
  • Strategic Planning
  • Basic Protocol 1: Building a Targeting Vector
  • Support Protocol 1: Linearization of the Completed Targeting Vector Prior to Transfection into Human Cells
  • Basic Protocol 2: Transfection of the Linearized Targeting Vector into Cultured Human Cells and Identification of Heterozygous Knockout Cells
  • Basic Protocol 3: Restoration of G418 Sensitivity Via Cre‐Mediated Excision of the neor Gene
  • Alternate Protocol 1: Swapping Selectable Markers in the Targeting Vector
  • Basic Protocol 4: Targeting of the Remaining Allele
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Building a Targeting Vector

  Materials
  • Genomic DNA (unit 2.2) derived from the parental cell line (e.g., HCT116; ATCC #CCL 247)
  • BAC DNA purification kit: e.g., Nucleobond Plasmid Maxi kit (Clontech) or similar kit from Qiagen (also see unit 2.1)
  • Yeast shuttle vectors (e.g., YEp24, pRS423, pRS424, pRS425, or pRS426, available from ATCC)
  • E. coli cells (DH10B)
  • pMC1neopolyA (Stratagene) or an IRES‐neor plasmid (available from John Sedivy at Brown University upon request)
  • EXPAND High Fidelity PCR System (Roche)
  • QIAX Gel Extraction Kit (Qiagen) or equivalent
  • Appropriate selective yeast media (Qbiogene)
  • Yeast PCR kit (Qbiogene)
  • Yeast RPM kit (Qbiogene)
  • Facility for BAC library screening (e.g., Research Genetics)
  • 30°C incubator
  • Additional reagents and equipment for primer design (unit 7.7), synthesis of oligonucleotides (unit 2.11), PCR (unit 15.1), gel purification of DNA fragments (unit 2.7), preparation and screening of a BAC library (http://www.chori.org/bacpac/home.htm), plating bacteria (unit 1.3), single‐cell PCR (unit 25.3), restriction digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), restriction mapping (units 3.2, 3.3 & 7.7), finding sequence information on the Web (units 19.2 & 19.3), dephosphorylation of DNA by calf intestinal phosphatase (unit 3.10), DNA subcloning and ligation (unit 3.16), introduction of plasmid DNA into E. coli (unit 1.8), colony hybridizations (unit 6.3), preparation of plasmid DNA (unit 1.7), DNA sequencing (Chapter 7), phenol extraction and ethanol precipitation of DNA (unit 2.5), gel purification of DNA fragments (units 2.5, 2.7, or 2.8), introduction of DNA into S. cerevisiae (unit 13.7), growth of S. cerevisiae (unit 13.2), and DNA preparation from S. cerevisiae (unit 13.11)

Support Protocol 1: Linearization of the Completed Targeting Vector Prior to Transfection into Human Cells

  Materials
  • Completed targeting vectors ( protocol 1)
  • TE buffer ( appendix 22)
  • Additional reagents and equipment for restriction digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), and phenol extraction and ethanol precipitation of DNA (unit 2.1)

Basic Protocol 2: Transfection of the Linearized Targeting Vector into Cultured Human Cells and Identification of Heterozygous Knockout Cells

  Materials
  • recipeComplete McCoy's 5A medium (see recipe) with and without 0.6 µg/ml G418 (geneticin, Life Technologies; see unit 9.5)
  • HCT116 cells (ATCC #CCL 247)
  • Lipofectamine reagent (Invitrogen; see unit 9.4)
  • Linearized targeting vector ( protocol 2)
  • Plasmid unrelated to targeting vector and lacking neor gene (negative control for colony formation)
  • PMC1neopolyA plasmid (Stratagene)
  • CMV β‐gal plasmid (Clontech)
  • Hanks' Balanced Salt Solution (HBSS, Life Technologies; also see appendix 22)
  • 0.05% (w/v) trypsin/0.53 mM tetrasodium EDTA (Life Technologies)
  • 25‐cm2 and 75‐cm2 tissue culture flasks
  • 8‐channel pipettor (e.g., Easy Step with 8‐port manifold from Continental Laboratory Products) with 50‐ml reservoir (Brinkmann 22‐49‐614‐0)
  • 96‐well tissue culture plates
  • 24‐well tissue culture plates
  • 15‐ml conical centrifuge tubes
  • Additional reagents and equipment for culture of mammalian cells ( appendix 3F), transfection of mammalian cells (unit 9.4), Xgal staining of cultured cells (unit 9.10), crystal violet staining of cultured cells (as for plaque assay; see unit 16.16, protocol 2), preparation of genomic DNA (unit 2.2), PCR (unit 15.1), and Southern blotting (unit 2.9)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 3: Restoration of G418 Sensitivity Via Cre‐Mediated Excision of the neor Gene

  Materials
  • High‐titer Cre‐expressing adenovirus (e.g., Microbix Biosystems)
  • Heterozygous knockout cell lines (see protocol 3) growing at 50% confluence in 25‐cm2 tissue culture flasks
  • recipeComplete McCoy's 5A medium (see recipe) with and without 0.6 µg/ml G418 (geneticin, Life Technologies; see unit 9.5)
  • Hanks' Balanced Salt Solution (HBSS, Life Technologies; also see appendix 22)
  • 0.05% (w/v) trypsin/0.53 mM tetrasodium EDTA (Life Technologies)
  • 25‐cm2 tissue culture flasks
  • 96‐well tissue culture plates
  • 24‐well tissue culture plates
  • Additional reagents and equipment for culture of mammalian cells (including counting and cryopreservation; appendix 3F), plating cells at limiting dilution (as for hybridoma cells; unit 11.8), and crystal violet staining of cultured cells (as for plaque assay; see unit 16.16, protocol 2)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.
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Figures

Videos

Literature Cited

Literature Cited
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