Glutamate Cysteine Ligase (GCL) Transgenic and Gene‐Targeted Mice for Controlling Glutathione Synthesis

Isaac Mohar1, Dianne Botta1, Collin C. White1, Lisa A. McConnachie1, Terrance J. Kavanagh1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.16
DOI:  10.1002/0471140856.tx0616s39
Online Posting Date:  February, 2009
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Abstract

The tripeptide glutathione (GSH) has important antioxidant properties, scavenges free radicals, and serves as a cofactor for glutathione S‐transferase conjugation of many xenobiotics. GSH is synthesized in two steps. The first and, often, rate‐limiting step is the formation of γ‐glutamylcysteine, which is catalyzed by the inducible heterodimeric enzyme glutamate cysteine ligase (GCL). The two subunits of GCL are the catalytic subunit (GCLC) and the modifier subunit (GCLM). In this unit, the generation and basic characterization methodologies of transgenic mouse models that have been developed to (1) conditionally over express both GCL subunits; (2) lack GCLM (Gclm null); and (3) create a hybrid between Gclm conditional over‐expressing mice on a Gclm null genetic background are discussed. These models can be used to explore the fundamental role of GCLC and GCLM in GSH synthesis, as well as the toxicological role of GSH and its synthesis in xenobiotic metabolism and response to oxidative stress. Curr. Protoc. Toxicol. 39:6.16.1‐6.16.20. © 2009 by John Wiley & Sons, Inc.

Keywords: glutathione; glutamate cysteine ligase; oxidative stress; transgenic mouse; genetic model

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Colony Management and Genotyping of GCLC and GCLM Transgenic Mice
  • Support Protocol 1: Induction of GCLC and GCLM Transgenes by Mifepristone Administration
  • Support Protocol 2: Administration of Mifepristone in Drinking Water to Induce GCLC and GCLM Transgenes
  • Basic Protocol 2: Determination of GCLC and GCLM mRNA Expression by Quantitative Real‐Time PCR
  • Basic Protocol 3: Analysis of GCLC and GCLM Protein Expression by Immunoblot
  • Basic Protocol 4: Determination of GCL Enzyme Activity
  • Alternate Protocol 1: Analysis of GCL Activity in Tissue Using a 96‐Well Plate
  • Basic Protocol 5: Measurement of Combined Glutathione and γ‐GC in Tissue
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Colony Management and Genotyping of GCLC and GCLM Transgenic Mice

  Materials
  • Breeding pairs of mice of optimal age (8 to 20 weeks old)
  • IACUC‐approved food and water
  • Tissue lysis buffer (for PCR; see recipe)
  • Proteinase K (see recipe)
  • DNase‐free water
  • Taq DNA polymerase and 10× buffer
  • DMSO
  • dNTPs (dATP, dTTP, dGTP, dCTP)
  • Primers (forward and reverse pairs; Table 6.16.3)
  • Genomic DNA (unknowns and positive controls)
  • 1% agarose gel in 0.5× TBE buffer
  • 0.5× TBE agarose electrophoresis buffer (from 5× stock)
  • CYBR Safe (Invitrogen cat. no. S33102) or ethidium bromide
  • 6× DNA sample buffer (see recipe)
  • IACUC‐approved animal housing facility
  • IACUC‐approved animal cages and bedding
  • Numbered mouse ear tags and tag crimper
  • Sterile scissors or ear puncher (Kent Scientfic Corp. no. INS50075)
  • 1.5‐ml microcentrifuge tubes, sterile
  • 55°C water bath
  • 95°C water bath or heating block
  • 0.5‐ml thin‐walled PCR tubes
  • Thermal cycler
  • Gel electrophoresis apparatus and power source
  • UV light box
  • Gel photography equipment

Support Protocol 1: Induction of GCLC and GCLM Transgenes by Mifepristone Administration

  Materials
  • Mice (of desired genotype(s), see Table 6.16.1)
  • Mifepristone (Sigma cat. no. M8046)
  • Sesame oil (Sigma)
  • Scale (to weigh mice)
  • 1‐ml syringe with 18‐ to 23‐G needle

Support Protocol 2: Administration of Mifepristone in Drinking Water to Induce GCLC and GCLM Transgenes

  • 25 µg/ml mifepristone in drinking water

Basic Protocol 2: Determination of GCLC and GCLM mRNA Expression by Quantitative Real‐Time PCR

  Materials
  • Liver tissue (fresh or stored at −80°C)
  • TRIzol (Invitrogen)
  • RNA isolation kit (e.g., Qiagen RNeasy isolation kit)
  • SuperScript cDNA synthesis kit (Invitrogen)
  • TaqMan probes for Gclc, Gclm, and Gapdh (see Table 6.16.5)
  • Tissue homogenizer or microtip sonicator
  • Additional reagents and equipment for real‐time PCR (unit 4.15)
    Table 6.6.5   MaterialsTaqMan Probes for Quantification of GclcTG and GclmTG cDNA

    Target cDNA (direction) Sequence (5′ to 3′) ca
    Gclc (forward) (6FAM)‐TTC TCC AGA TGC TCT CTT CT‐(MGB)
    Gclm (forward) (6FAM)‐CAC AAT GAC CCG AAA GAA‐(MGB)
    Transgene (reverse) GTG CTG GAT ATC TGC AGA ATT CG
    GAPDH (forward) (6FAM)‐CAC TCA TGA CCA CAG TCC ATG CCA TCA C‐(TAMRA)
    GAPDH (reverse) GAG GGG CCA TCC ACA GTC TT

     c6FAM is a fluorescent reporter dye at the 5′ end of the oligonucleotide. MGB is used to enhance probe binding and is 3′ of a non‐fluorescent quencher. TAMRA is a quenching dye. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is often used to standardize RT‐PCR results.

Basic Protocol 3: Analysis of GCLC and GCLM Protein Expression by Immunoblot

  Materials
  • Tissue sample
  • Homogenization buffer: protein stabilization buffer such as TES/SB containing protease inhibitor (e.g., Complete Mini Protease Inhibitor cocktail, Roche cat. no. 11836153001)
  • Polyacrylamide gel (10%, 12%, or 4% to 20%; precast gels from Bio‐Rad or Invitrogen)
  • 1× SDS PAGE electrophoresis buffer (see recipe)
  • 1× native PAGE buffer, pre‐chilled (see recipe)
  • 4× SDS protein sample buffer (see recipe)
  • 6× native protein sample buffer (see recipe)
  • Anti‐mouse GCLC antibody (polyclonal rabbit) or anti‐mouse GCLM antibody (polyclonal rabbit) (available commercially; also from T.J. Kavanagh, University of Washington)
  • Blocking solution: PBS‐T buffer (PBS + 0.1% Tween‐20) + 5% (w/v) non‐fat milk powder
  • PBS‐T buffer: PBS + 0.1% Tween‐20
  • Anti‐rabbit HRP‐conjugated antibody (polyclonal goat; such as Upstate cat. no. 12‐348)
  • PBS ( appendix 2A)
  • Electrophoresis apparatus and power source
  • PVDF or nitrocellulose membranes
  • X‐ray film

Basic Protocol 4: Determination of GCL Enzyme Activity

  Materials
  • Tissue sample
  • Homogenization buffer: TES/SB buffer (see recipe)
  • Protease inhibitors (protease inhibitor cocktail, e.g., Roche Complete cat. no. 11836153001)
  • GCL reaction cocktail (for NDA method; see recipe)
  • 5 mM L‐cysteine in homogenization buffer
  • 200 mM 5‐sulfosalicylic acid (SSA; Sigma)
  • GSH standards
  • 0.2 M NEM/0.02 M KOH solution
  • 10 mM Tris(2‐carboxyethyl)‐phosphine in water (TCEP; Pierce cat. no. 20490)
  • 10 mM naphthalenedicarboxaldehyde in DMSO (NDA; Aldrich cat. no. 382019)
  • 50 mM Tris⋅Cl, pH 10 ( appendix 2A)
  • 0.5 N NaOH
  • Centrifuge tubes
  • Centrifuge (swinging‐bucket) with 96‐well plate adaptor
  • 96‐well plates, round bottom (Costar no. 3799)
  • 37°C water bath or incubator suitable for 96‐well plates
  • 96‐well plates, black polystyrene (Costar no. 3916)
  • UV 96‐well plate reader

Alternate Protocol 1: Analysis of GCL Activity in Tissue Using a 96‐Well Plate

  Materials
  • 10% (w/v) 5‐sulfosalicylic acid (SSA; Sigma)
  • Tissue (fresh or stored at −80°C) or cell homogenate
  • TES/SB tissue homogenization buffer (see recipe)
  • Glutathione, reduced (GSH; γ–glutamylcysteinylglycine; Sigma)
  • 10 mM Tris(2‐carboxyethyl)‐phosphine in water (TCEP; Pierce)
  • 10 mM naphthalenedicarboxaldehyde in DMSO (NDA; Sigma)
  • 0.2 M N‐ethylmorpholine (NEM; Sigma)/0.02 M KOH
  • 0.5 N NaOH
  • 96‐well (black) plates suitable for fluorescence (Costar no. 3916)
  • UV 96‐well plate reader
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Figures

Videos

Literature Cited

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Key References
   Botta et al., 2006. See above.
  This study provides experimental evidence for the mifepristone‐mediated induction of the GclcTG/GclmTG/GLVP mice and the mediated protection against acetaminophen‐induced liver damage.
   Botta et al., 2008. See above.
  This study provides a review of GCL transgenic mice and presents the effect of mifepristone‐mediated induction of the GclmTG/GLVP/Gclm null model and susceptibility to acetaminophen‐induced liver damage.
   McConnachie et al., 2007. See above.
  This study provides a further description of the Gclm null model and the effect on susceptibility to acetaminophen‐induced liver damage.
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