Modification of Bacterial Artificial Chromosomes (BACs) and Preparation of Intact BAC DNA for Generation of Transgenic Mice

Shiaoching Gong1, X. William Yang2

1 Rockefeller University, New York, New York, 2 Department of Psychiatry and Biobehavioral Sciences, Center for Neurobehavioral Genetics, Neuropsychiatric Institute, David Geffen School of Medicine at UCLA, Los Angeles, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.21
DOI:  10.1002/0471142301.ns0521s31
Online Posting Date:  May, 2005
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Abstract

BAC transgenesis is a powerful tool for the study of gene expression and gene function in the mouse in vivo. In this unit, detailed protocols are provided for modification (i.e., marker gene insertion, deletion, or point mutation) of BACs by homologous recombination in E. coli. This method utilizes a shuttle vector that allows transient expression of the E. coli RecA gene to support homologous recombination in the BAC host bacteria. In addition, two protocols are provided for purification of BAC DNA for microinjection to generate transgenic mice. Since BAC DNA is prone to degradation, which may introduce positional effects in transgenic mice, two methods are given for purification of intact BAC DNA for subsequent microinjection.

     
 
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Table of Contents

  • Basic Protocol 1: Modification of BACs Using the pLD53.SC‐AB Shuttle Vector
  • Alternate Protocol 1: Preparation of BAC DNA by Alkaline Lysis and Sepharose CL‐4B Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Modification of BACs Using the pLD53.SC‐AB Shuttle Vector

  Materials
  • pLD53.SC‐AB shuttle vector (Available from N. Heintz, Rockefeller University; Email: )
  • Pir2 competent cells
  • Plasmid DNA isolation kit (miniprep and midiprep kits, Qiagen) containing:
    • Buffer P1
    • Buffer P2
    • Buffer P3
  • Asc I restriction enzyme (New England Biolabs)
  • Not I restriction enzyme (New England Biolabs)
  • Shrimp alkaline phosphatase (SAP; Roche Diagnostics)
  • SeaPlaque low‐melting agarose (Cambrex)
  • DNA gel purification kit (e.g., Geneclean, Bio101)
  • High DNA mass ladder (Invitrogen)
  • BAC clone (e.g., BACPAC Resource Center at Oakland Children's Hospital)
  • DNA ligation kit (e.g., Version 2, Takara)
  • LB/Amp plates ( appendix 2A)
  • Alkaline lysis solution (see recipe)
  • Luria broth (LB) medium ( appendix 2A)
  • 10% (v/v) glycerol, cold
  • Liquid nitrogen or 100% ethanol
  • SOC medium (BD Diagnostics)
  • Ampicillin (Amp) and chloramphenicol (Chl)
  • LB/Chl medium: LB ( appendix 2A) containing 20 µg/ml Chl
  • LB/Chl/Amp medium: LB ( appendix 2A) containing 20 µg/ml Chl and 30 µg/ml Amp
  • LB/Chl/sucrose plates: LB‐agar plates supplemented with 20 µg/ml Chl and 5% sucrose
  • LB/Chl plate: LB‐agar plates supplemented with 20 µg/ml Chl
  • LB/Chl/Amp plates: LB‐agar plates supplemented with 20 µg/ml Chl and 30 µg/ml Amp
  • 10 mM EDTA, pH 8.0
  • 2 M potassium acetate (see recipe), cold
  • 100% isopropanol
  • TE buffer (see recipe)
  • 70% and 100% ethanol
  • Cesium chloride (CsCl)
  • 10 mg/ml ethidium bromide
  • NaCl‐saturated butanol (see recipe)
  • Embryo transfer water (Sigma)
  • 3 M sodium acetate
  • PI‐Sce I enzyme and 10× buffer (New England Biolabs)
  • Injection buffer (see recipe)
  • 50‐ml centrifuge tubes
  • Electroporator (Micropulser, BioRad)
  • 0.1‐cm electroporator cuvette
  • 17 × 100–mm polypropylene round‐bottom tube
  • Incubator shaker
  • Pulsed‐field gel system
  • 250‐ and 500‐ml bottles
  • 5/8 × 3–in. Beckman Quickseal tube
  • 10‐ml syringe
  • 18‐ and 23‐G needles
  • Ultracentrifuge and NVT65 rotor (Beckman)
  • 2.0‐ml microcentrifuge tubes
  • 25‐mm, 0.025‐µm filters (Millipore)
  • 100‐mm petri dishes
  • Kodak Gel Logic 100 imaging system
CAUTION: Ethidium bromide is a mutagen and environmental hazard. It should be handled carefully with gloves, and disposed of properly. Methods of disposal vary between different institutions. Consult the institution's environmental safety office for the preferred means of storage and disposal of ethidium waste.

Alternate Protocol 1: Preparation of BAC DNA by Alkaline Lysis and Sepharose CL‐4B Chromatography

  • Freshly streaked LB/Chl plates (see protocol 1)
  • 100% isopropanol (Sigma)
  • 1:1 (v/v) phenol (pH 8.0)/chloroform
  • Sepharose CL‐4B resin
  • 10× DNA dye
  • Ethidium bromide: 30 µl of 10 mg/ml stock in 500 ml water
  • 70‐µm cell strainer
  • Vacuum
  • 30‐ml centrifuge tubes
  • 5‐ml sterile plastic pipet (Falcon)
  • 24‐well plate
  • Parafilm
CAUTION: Ethidium bromide is a mutagen and environmental hazard. It should be handled carefully with gloves, and disposed of properly. Methods of disposal vary between different institutions. Consult the institution's environmental safety office for the preferred means of storage and disposal of ethidium waste.
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Figures

Videos

Literature Cited

Literature Cited
   Antoch, M.P., Song, E.J., Chang, A.M., Vitaterna, M.H., Zhao, Y., Wilsbacher, L.D., Sangoram, A.M., King, D.P., Pinto, L.H., and Takahashi, J.S. 1997. Functional identification of the mouse circadian clock gene by transgenic BAC rescue. Cell 89:655‐667.
   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.
   Gong, S., Yang, X.W., Li, J., and Heintz, N. 2002. Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kγ origin of replication. Genome Res. 12:1992‐1998.
   Gong, S., Zheng, C, Goughty, 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.
   Heintz, N. 2001. BAC to the future: The use of BAC transgenic mice for neuroscience research. Nat. Rev. Neurosci. 2:861‐870.
   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.
   McPherson J.D. et al. 2001. A physical map of the human genome. Nature 409:934‐941.
   Shizuya, H., Birren, B., Kim, U.J., Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M. 1992. Cloning and stable maintenance of 300‐kilobase‐pair fragments of human DNA in Escherichia coli using an F‐factor‐based vector. Proc. Natl. Acad. Sci. U.S.A. 89:8794‐8797.
   Valenzuela, D.M., Murphy, A.J., Frendewey, D., Gale, N.W., Economides, A.N., Auerbach, W., Poueymirous, W.T., Adams, N.C., Rojas, J., Yasenchak, J., Chernomorsky, R., Boucher, M., Elsasser, A.L., Esau, L., Zheng, J., Griffiths, J.A., Wang, X., Su, H., Xue, Y., Dominguez, M.G., Noguera, I., Torres, R., Macdonald, L.E., Stewart, A.F., DeChiara, T.M., and Yancopoulos, G.D. 2003. High‐throughput engineering of the mouse genome coupled with high‐resolution expression analysis. Nat. Biotechnol. 6:652‐659.
   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.
   Yang, X.W., Wynder, C., Doughty, M.L., and Heintz, N. 1999. BAC‐mediated gene‐dosage analysis reveals a role for Zipro1 (Ru49/Zfp38) in progenitor cell proliferation in cerebellum and skin. Nat. Genet. 22:327‐335.
   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.
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