Reporter Genes to Detect Cre Excision in Mice

Corrine P. Lobe1, Caiying Guo1

1 Sunnybrook and Womens College Health Sciences Centre and the University of Toronto, Ontario, Canada
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 15.4
DOI:  10.1002/0471140856.tx1504s10
Online Posting Date:  February, 2002
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Abstract

Transgenic mouse lines that express Cre recombinase with tissue and temporal specificity are being developed as important new tools to manipulate the mouse genome.

     
 
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Table of Contents

  • Basic Protocol 1: Analyzing Whole‐Mount Embryos or Tissues for Reporter Gene Expression
  • Basic Protocol 2: Analyzing Tissue Sections for Reporter Gene Expression
  • Support Protocol 1: Genotyping Cre Reporter Mice and Cre Transgenic Mice by PCR
  • Support Protocol 2: Genotyping Cre Reporter Mice and Cre Transgenic Mice by lacZ Expression
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Analyzing Whole‐Mount Embryos or Tissues for Reporter Gene Expression

  Materials
  • Dissected Cre reporter double‐transgenic mouse embryos or tissues (see protocol 3Support Protocols 1 and protocol 42 for genotyping)
  • PBS ( appendix 2A), ice cold
  • 100 mM sodium phosphate buffer, pH 7.3 (see recipe), optional
  • lacZ fixative (see recipe), ice cold
  • 2% (w/v) paraformaldehyde/0.2% (v/v) glutaraldehyde in PBS, for large (≥E9.5) embryos only, ice cold
  • lacZ wash buffer (see recipe), ice cold
  • lacZ staining solution (see recipe)
  • AP wash buffer (see recipe)
  • AP staining solution (see recipe)
  • 0.1% (v/v) Tween 20/2 mM MgCl 2 in PBS
  • Petri dishes
  • 24‐well and 6‐well tissue culture plates
  • Dissecting microscope with light source and filter set for GFP visualization (Leica or BLS‐Ltd.)
  • 15‐ or 50‐ml snap‐cap tubes (e.g., Falcon), for en masse hPLAP staining
  • 70° to 75°C water bath
CAUTION: Paraformaldehyde and glutaraldehyde are hazardous chemicals; follow appropriate precautions for handling, storage, and disposal.NOTE: Signals will be best if initial tissue fixation and washing is done in ice‐cold solutions on ice and with gentle shaking.

Basic Protocol 2: Analyzing Tissue Sections for Reporter Gene Expression

  Materials
  • Dissected Cre reporter double‐transgenic mouse embryos or tissues (see Support Protocols protocol 31 and protocol 42 for genotyping)
  • PBS ( appendix 2A), ice cold
  • Tissue‐Tek OCT
  • 15% and 30% (w/v) sucrose in PBS, 4°C
  • 0.2% glutaraldehyde (v/v) in PBS, 4°C
  • lacZ wash buffer (see recipe), 4°C
  • lacZ staining solution (see recipe)
  • AP wash buffer (see recipe)
  • AP staining solution (see recipe)
  • 0.1% (v/v) Tween 20/2 mM MgCl 2 in PBS
  • Nuclear fast red stain: 5% (w/v) aluminum sulfate/0.1% (w/v) fast red (optional)
  • 70% and 90% (v/v) ethanol in PBS
  • 100% ethanol
  • 1:1 (v/v) ethanol/xylene
  • Xylene
  • Mounting solution (e.g., Cytoseal; Fisher)
  • Petri dishes
  • Coverslips
  • Compound microscope with light source and filter set for GFP visualization (Leica or BLS‐Ltd.)
  • Plastic molds for cryofreezing
  • Box for storing embedded samples, optional
  • Cryostat, −20°C
  • Poly‐lysine coated slides (e.g., Fisher)
  • Slide box
  • Staining jars
  • 70° to 75°C water bath
  • Light‐protected container, such as a plastic food container wrapped in foil
  • Additional reagents and equipment for fixing samples (see protocol 1)
CAUTION: Glutaraldehyde and xylene are hazardous chemicals; follow appropriate precautions for handling, storage, and disposal.NOTE: Signals will be best if initial tissue fixation and washing is done in ice‐cold solutions on ice and with gentle shaking.

Support Protocol 1: Genotyping Cre Reporter Mice and Cre Transgenic Mice by PCR

  Materials
  • Yolk sac, tail biopsy, or ear punch from potential transgenic mouse
  • PBS ( appendix 2A)
  • 1× proteinase K buffer (see recipe)
  • Mineral oil
  • dNTP cocktail: 2 mM each dATP, dCTP, dGTP, dTTP
  • Taq DNA polymerase
  • 10× PCR buffer ( appendix 3C)
  • 10 µM PCR primer for Cre recombinase:
    • 5′‐ATG TCC AAT TTA CTG ACC CC‐3′
    • 5′‐CGC CGC ATA ACC AGT GAA AC‐3′
  • 50°C incubator
  • PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis ( appendix 3A)

Support Protocol 2: Genotyping Cre Reporter Mice and Cre Transgenic Mice by lacZ Expression

  Materials
  • Ear punch from potential transgenic mouse
  • PBS ( appendix 2A)
  • 0.2% (v/v) glutaraldehyde in PBS
  • lacZ staining solution (see recipe)
  • 96‐well plate
  • 37°C incubator, optional
  • Dissecting microscope
CAUTION: Glutaraldehyde is a hazardous chemical; follow appropriate precautions for handling, storage, and disposal.
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Figures

Videos

Literature Cited

Literature Cited
   Akagi, K., Sandig, V., Vooijs, M., Van Der Valk, M., Giovannini, M., Strauss, M., and Berns, A. 1997. Cre‐mediated somatic site‐specific recombination in mice. Nucl. Acids Res. 25:1766‐1773.
   Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and Chambon, P. 1996. Ligand‐activated site‐specific recombination in mice. Proc. Natl. Acad. Sci. U.S.A. 93:10887‐10890.
   Furth, P.A., St. Onge, L., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., and Hennighausen, L. 1994. Temporal control of gene expression in transgenic mice by a tetracycline‐responsive promoter. Proc. Natl. Acad. Sci. U.S.A. 91:9302‐9306.
   Hebert, J.M. and McConnell, S.K. 2000. Targeting of cre to the foxg1 (BF‐1) locus mediates loxP recombination in the telencephalon and other developing head structures. Dev. Biol. 222:296‐306.
   Hoess, R.H., Wierzbicki, A., and Abremski, K. 1986. The role of the loxP spacer region in P1 site‐specific recombination. Nucl. Acids Res. 14:2287‐2300.
   Kawamoto, S., Niwa, H., Tashiro, F., Sano, S., Kondoh, G., Takeda, J., Tabayashi, K., and Miyazaki, J. 2000. A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre‐mediated recombination. FEBS Lett. 470:263‐268.
   Kellendonk, C., Tronche, F., Monaghan, A.‐P., Angrand, P.‐O., Stewart, F., and Schutz, G. 1996. Regulation of Cre recombinase activity by the synthetic steroid RU 486. Nucl. Acids Res. 24:1404‐1411.
   Lobe, C.G. and Nagy, A. 1998. Conditional genome alteration in mice. BioEssays 20:200‐208.
   Lobe, C.G., Koop, K.E., Kreppner, W., Lomeli, H., Gertsenstein, M., and Nagy, A. 1999. Z/AP, a double reporter for cre‐mediated recombination. Dev. Biol. 208:281‐292.
   Mao, X., Fujiwara, Y., and Orkin, S.H. 1999. Improved reporter strain for monitoring Cre recombinase‐mediated DNA excisions in mice. Proc. Natl. Acad. Sci. U.S.A. 96:5037‐5042.
   Novak, A., Guo, C., Yang, W., Nagy, A., and Lobe, C.G. 2000. Z/EG, a double reporter transgenic mouse line that expresses enhanced Green Fluorescent Protein upon Cre‐mediated recombination. Genesis 28:147‐155.
   Rivera, V.M., Clackson, T., Natesan, S., Pollock, R., Amara, J.F., Keenan, T., Magari, S.R., Phillips, T., Courage, N.L., Cerasoli, F., Holt, D.A., and Gilman, M. 1996. A humanized system for pharmacological control of gene‐expression. Nature Med. 2:1028‐1032.
   Sauer, B. 1998. Inducible gene targeting in mice using the Cre/lox system. Methods 14:381‐392.
   Soriano, P. 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain [letter]. Nature Genet. 21:70‐71.
   Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503‐517.
   Sternberg, N. and Hamilton, D. 1981. Bacteriophage P1 site‐specific recombination I. Recombination between loxP sites. J. Mol. Biol. 150:467‐486.
   Tsien, J., Chen, D., Gerber, D., Tom, C., Mercer, E., Anderson, D., Mayford, M., Kandel, E., and Tonegawa, S. 1996. Subregion‐ and cell type‐restricted gene knockout in mouse brain. Cell 87:1317‐1326.
Internet Resources
   http://www.mshri.on.ca/develop/nagy/Cre.htm
   This website lists Cre transgenic lines, available from laboratories, that have widespread tissue‐specific or inducible Cre activity.
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