Recombineering‐Based Procedure for Creating Cre/loxP Conditional Knockouts in the Mouse

Jason Bouvier1, Jr‐Gang Cheng2

1 Tetracore, Inc., Rockville, Maryland, 2 UNC‐Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 23.13
DOI:  10.1002/0471142727.mb2313s85
Online Posting Date:  January, 2009
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Abstract

Gene targeting in the mouse is an essential tool for studying gene function and creating models of human disease. The method described in this unit takes advantage of bacterial artificial chromosomes, Cre/loxP and FLPe/FRT systems, and recently evolved recombineering approaches to simplify the preparation of targeting constructs for generation of conditional knockout (CKO) animals. This method has been used to generate >30 CKO constructs, most of them successfully used to target mouse ES cells and establish mutant mice. Design and preparation of the CKO construct, as well as step‐wise troubleshooting guidelines, are described in detail. Curr. Protoc. Mol. Biol. 85:23.13.1‐23.13.27. © 2009 by John Wiley & Sons, Inc.

Keywords: gene targeting; conditional knockout; recombineering; mouse; Cre/loxP

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Manipulating DNA with Recombineering
  • Basic Protocol 2: Quality Control for Recombineering
  • Alternate Protocol 1: Adding a Gene‐Trap Cassette
  • Support Protocol 1: Making Electrocompetent Cells
  • Support Protocol 2: Electroporation into DH10B and Its Derivatives
  • Support Protocol 3: Working with Recombinogenic Bacterial Strains
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Manipulating DNA with Recombineering

  Materials
  • Low‐salt LB medium and agar plates (unit 1.1; 1.0% tryptone, 0.5% yeast extract, 0.5% NaCl) with the following antibiotics and additives as necessary:
    • 20 µg/ml chloramphenicol (34 mg/ml in ethanol stock)
    • 6.25 µg/ml tetracycline (12.5 mg/ml in ethanol stock; store in dark)
    • 100 µg/ml ampicillin (50 mg/ml in water stock)
    • 25 µg/ml kanamycin (50 mg/ml in water stock)
    • 1 M isopropylthio‐β‐D‐galactoside (IPTG; 20 µl/plate; optional for galK selection)
    • 50 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside in DMFO (X‐gal, 50 µl/plate; optional for galK selection)
  • BAC containing gene of interest (see ), as glycerol stock or culture plates
  • Miniprep kit (e.g., Qiagen)
  • Isopropanol
  • 70% ethanol
  • 10 mM Tris⋅Cl buffer, pH 8 ( appendix 22)
  • Recombinogenic E. coli strains:
    • DY380 (or galK SW102): DH10B derived, contains recombination proteins exo, bet, and gam, controlled by temperature‐sensitive repressor, and Tet+
    • EL350 (or galK SW106): Tet+ replaced with arabinose‐inducible Cre gene, for recombination between two loxP sites
  • PCR primers and reagents (see Tables 23.13.1, 23.13.2, and 23.13.3)
  • Taq DNA polymerase with 10× Thermopol buffer (NEB) or Phusion high‐fidelity Taq DNA polymerase with 5× HF buffer (Finnzymes) or similar high‐fidelity enzyme
  • PCR purification kit (e.g., Qiagen)
  • Restriction endonucleases: EcoRI, XmaI, XhoI, BamHI, XbaI, SmaI
  • Gel extraction kit (e.g., Qiagen)
  • Vector DNA:
    • pBS‐DT: gene‐targeting vector that contains the negative selection marker diphtheria toxin (DT), a polypeptide chain under the regulation of a Pol II promoter cloned into the SmaI site of pBluescript KSII vector
    • pL451: contains duet promoters neo cassette flanked by two FRT sites and one loxP site in pBluescriptKS
    • pL452: contains duet promoters neo cassette flanked by two loxP sites in pBluescriptKS
  • Optional DNA constructs:
    • λ‐RED in a plasmid: pKD46
    • Recombinase in a plasmid under the control of arabinose: pBAD‐Cre, pBAD‐FLPe
  • Rapid DNA ligation kit (e.g., Roche)
  • T4 DNA ligase with associated buffer (e.g., Promega)
  • Competent E. coli strain for cloning, e.g., JM109, DH10B, DH5α, or Stb2 (optional, Invitrogen)
  • SOC medium (unit 1.8)
  • 10% arabinose in water
  • 14‐ and 50‐ml conical tubes
  • 32°C orbital shaking incubator
  • Tabletop centrifuge
  • 1.5‐ and 2.0‐ml microcentrifuge tubes
  • Thermal cycler
  • 42°C shaking water bath
  • Spectrophotometer with 1‐ml disposable cuvettes
  • Electroporator (e.g., Eppendorf Electroporator 2510 with 1‐mm electroporation cuvettes)
  • 125‐ and 250‐ml Erlenmeyer flasks
  • Additional reagents and equipment for BAC minipreps (e.g., unit 5.9), electroporation (see Support Protocols protocol 41 and protocol 52; unit 9.3), PCR (unit 15.1), agarose gel electrophoresis (unit 2.5), restriction endonuclease digestion (unit 3.1), gel purification (unit 2.6), and DNA ligation (units 3.14& 3.16)
NOTE: Basic molecular methods can be carried out using commercially available kits (e.g., for minipreps, purification of PCR products, gel‐purification of DNA fragments, and ligation) or using standard protocols cited here.NOTE: All cultures are grown at 32°C. Liquid cultures are grown at 32°C in an orbital shaking incubator.

Basic Protocol 2: Quality Control for Recombineering

  Materials
  • Recombinogenic E. coli strains:
    • EL250 (or galK SW105): Tet+ replaced with arabinose‐inducible FLPe gene, for recombination between two FRT sites
    • EL350 (or galK SW106): Tet+ replaced with arabinose‐inducible Cre gene, for recombination between two loxP sites
  • Low‐salt LB medium and agar plates (unit 1.1; 1.0% tryptone, 0.5% yeast extract, 0.5% NaCl) with the following antibiotic and additive as necessary:
    • 100 µg/ml ampicillin (50 mg/ml stock in water)
    • 25 µg/ml kanamycin (50 mg/ml stock in water)
    • 0.1% L‐arabinose (10% stock in water; Sigma)
  • CKO targeting construct (see protocol 1)
  • SOC medium (unit 1.8)
  • PCR primers and reagents (see Tables 23.13.1, 23.13.2, and 23.13.3)
  • 14‐ml conical tubes
  • 32°C orbital shaker incubator
  • 250‐ml Erlenmeyer flasks
  • Electroporator (e.g., Eppendorf Electroporator 2510 with 1‐mm electroporation cuvettes)
  • Spectrophotometer with 1‐ml disposable cuvettes
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for electroporation (see Support Protocols protocol 41 and protocol 52; unit 9.3), PCR (unit 15.1), and agarose gel electrophoresis (unit 2.5)

Alternate Protocol 1: Adding a Gene‐Trap Cassette

  • Plasmid containing a gene‐trap cassette (splicing acceptor, tracer, selection marker) floxed with FRT sites or a negative selection marker (SacB); e.g., pSA‐I‐Venus‐451‐SacB or P15A‐FRT‐Geo‐FRT‐loxP
  • 50% sucrose

Support Protocol 1: Making Electrocompetent Cells

  Materials
  • 10% glycerol
  • Liquid bacterial culture
  • 2‐ml microcentrifuge tubes
  • 50‐ml centrifuge tubes
  • Tabletop centrifuge

Support Protocol 2: Electroporation into DH10B and Its Derivatives

  Materials
  • Electrocompetent cells (see protocol 4)
  • DNA (e.g., freshly prepared BAC, plasmid, or targeting cassette)
  • SOC medium
  • LB agarose plates containing the appropriate antibiotics
  • 1‐mm electroporation cuvettes
  • 1.5‐ml microcentrifuge tubes
  • Electroporator (e.g., Eppendorf)
  • 32°C orbital shaking incubator

Support Protocol 3: Working with Recombinogenic Bacterial Strains

  Materials
  • Bacterial colonies grown on plates
  • LB medium
  • 10% glycerol or 7% DMSO
  • 5‐ml transparent tubes (e.g., Falcon)
  • 32° and 42°C incubators
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Figures

Videos

Literature Cited

   Copeland, N.G., Jenkins, N.A., and Court, D.L. 2001. Recombineering: A powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2:769‐779
   Datsenko, K.A. and Wanner, B.L. 2000. One‐step inactivation of chromosomal genes in Escherichia coli K‐12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97:6640‐6645.
   Gong, S., Zheng, C., Doughty, M.L., Losos, K., Didkovsky, N., Schambra, U.B., Nowak, N.J., Joyner, A., Leblanc, G., Hatten, M.E., and Heintz, N. 2003. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917‐925.
   Lee, E.C., Yu, D., Martinez de Velasco, J., Tessarollo, L., Swing, D.A., Court, D.L., Jenkins, N.A., and Copeland, N.G. 2001. A highly efficient Escherichia coli–based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56‐65.
   Lewandoski, M. 2001. Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2:743‐755.
   Liu, P., Jenkins, N.A., and Copeland, N.G. 2003. A highly efficient recombineering‐based method for generating conditional knockout mutations. Genome Res. 13:476‐484.
   Warming, S., Costantino, N., Court, D.L., Jenkins, N.A., and Copeland, N.G. 2005. Simple and highly efficient BAC recombineering using galK selection. Nucl. Acids Res. 33:e36.
   Yang, X.W., Model, P., and Heintz, N. 1997. Homologous recombination based modification in Escherichia coli. and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15:859‐865.
   Zhang, Y., Buchholz, F., Muyrers, J.P., and Stewart, A.F. 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20:123‐128.
Internet Resources
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