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Use of Bacterial Artificial Chromosomes in Generating Targeted Mutations in Human and Mouse Cytomegaloviruses

Eva Maria Borst1,  Corinna Benkartek1,  Martin Messerle1

1Institute of Virology, Hannover Medical School, Hannover, Germany

Publication Name: 
Current Protocols in Immunology
Unit Number: 
Unit 10.32
DOI: 
10.1002/0471142735.im1032s77
Online Posting Date: 
May, 2007
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Abstract

Cloning of cytomegalovirus (CMV) genomes as bacterial artificial chromosomes (BAC) in E. coli and their manipulation using the techniques of bacterial genetics has greatly facilitated the construction of CMV mutants. This unit describes easily applicable procedures that allow rapid introduction of any kind of targeted mutation into BAC-cloned CMV genomes. Protocols for the reconstitution of virus from isolated BAC DNA, preparation of a virus stock, and isolation and characterization of viral DNA are also included. Special emphasis is laid on description of critical steps and thorough characterization of the altered BACs.

Keywords: cytomegaloviruses; bacterial artificial chromosomes; mutagenesis

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Mutagenesis of CMV BACS Using Linear DNA Fragments and the Recombination Enzymes of Bacteriophage
  • Alternate Protocol 1: Manipulation of CMV BACS Using the galK Selection Marker
  • Alternate Protocol 2: Construction of CMV BAC Mutants by Two-Step Allele Replacement (Shuttle Mutagenesis)
  • Support Protocol 1: Excision of the Selection Marker from Mutated CMV BACS by Flp Recombinase
  • Support Protocol 2: Insertion of Specific Sequences into a CMV BAC Using Flp-Mediated Recombination
  • Support Protocol 3: Reconstitution of CMV Mutants by Transfection into Permissive Cells
  • Support Protocol 4: Isolation of Viral DNA From Virus Particles of the CMV Mutant and Analysis of the Genomic Structure by Restriction Analysis and Southern Blotting
  • Support Protocol 5: Large Scale Preparation of hCMV Mutants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Mutagenesis of CMV BACS Using Linear DNA Fragments and the Recombination Enzymes of Bacteriophage

 Materials
  • LB medium with and without 17 µg/ml chloramphenicol (see recipe)
  • CMV BAC–containing E. coli strain (e.g., strain DH10B), typically recombination-incompetent (recA): obtain from researchers generating the CMV BAC of interest
  • Double-distilled (dd) H2O, sterile and ice-cold
  • 10% (v/v) glycerol, sterile and ice-cold
  • Plasmid expressing the recombination genes red , , (e.g., pKD46; Datsenko and Wanner, 2000): available from the E. coli Genetic Stock Center at Yale University (http://cgsc.biology.yale.edu)
  • LB agar plates containing 17 µg/ml chloramphenicol and 50 µg/ml ampicillin (see recipe)
  • LB medium containing 17 µg/ml chloramphenicol and 50 µg/ml ampicillin (see recipe)
  • 10% (w/v) l-(+)-arabinose (Sigma)
  • Plasmid encoding an antibiotic resistance (e.g., kanamycin resistance), ideally flanked by FRT sites, and based on a conditional replicon (e.g., oriR6K): e.g., pKD4 (Datsenko and Wanner, 2000) or pGP704-Kan (Borst and Messerle, 2005)
  • Oligonucleotide primers for antibiotic resistance plasmid
  • Taq polymerase with proofreading activity (e.g., Phusion high-fidelity DNA polymerase; NEB)
  • TE buffer: 10 mM Tris×Cl pH 8.0 (appendix 2A)/10 mM EDTA (see appendix 2A)
  • LB agar plates containing 17 µg/ml chloramphenicol and 25 µg/ml kanamycin (see recipe)
  • LB medium containing 17 µg/ml chloramphenicol and 25 µg/ml kanamycin (see recipe)
  • GTE-solution (see unit 10.3)
  • NaOH/SDS solution: 0.2 M NaOH/1% SDS; prepare just before use
  • 3 M potassium acetate pH 4.8 (see unit 10.3), ice-cold
  • Isopropanol
  • 70% Ethanol
  • 20 µg/ml RNase A/TE buffer (see recipe)
  • Appropriate restriction enzyme and 10× buffer
  • TBE buffer (unit 10.4)
  • 10× DNA loading buffer (unit 10.4)
  • Agarose
  • 1% ethidium bromide solution (unit 10.4)
  • 37°C incubator with shaker
  • 2-ml and 1.5-ml microcentrifuge tubes
  • Microcentrifuge
  • 30°C, 37°C, and 43°C incubators
  • 250-ml conical flask
  • Thermal cycler
  • Spin column purification kit (e.g., QIAquick PCR purification kit; Qiagen)
  • Gel electrophoresis chamber for agarose gels, ~23 × 40 cm
  • 12-well gel comb
  • Anion exchange chromatography kit (e.g., Nucleobond PC100; Macherey-Nagel)
  • Additional reagents and equipment for transforming bacteria by electroporation (Seidman et al., 1997 or appendix 3N), performing PCR (see unit 10.20), and analyzing DNA by restriction enzyme digestion (unit 10.8) and agarose gel electrophoresis (unit 10.4)

Alternate Protocol 1: Manipulation of CMV BACS Using the galK Selection Marker

 Materials
  • High-fidelity Taq polymerase
  • Plasmid containing a galK cassette (e.g., pgalK: galactokinase gene driven by the em7 promoter cloned on a pBluescript backbone; Warming et al., 2005)
  • DpnI and 10× digestion buffer (NEB)
  • TE (Tris EDTA) buffer (appendix 2A)
  • E. coli strain SW102 (Warming et al., 2005) containing the CMV BAC
  • 1% agarose gel (see unit 10.4)
  • LB medium with and without 12.5 µg/ml chloramphenicol (see recipe)
  • E. coli strain SW102 (Warming et al., 2005) containing the CMV BAC (see Warming et al., 2005)
  • M9 medium (see recipe)
  • M63 galactose minimal medium agar plates (see recipe)
  • MacConkey agar plates, containing 1% (v/v) galactose and 12.5 µg/ml chloramphenicol (see recipe)
  • M63 DOG minimal medium agar plates (see recipe)
  • Spin column purification kit (QIAquick PCR purification kit; Qiagen)
  • 32°C shaking water bath, incubator, or thermomixer
  • 50-ml conical flask
  • 42°C shaking water bath
  • Additional reagents and equipment for performing PCR (see unit 10.20), analyzing DNA by agarose gel electrophoresis (unit 10.4), and transforming bacteria by electroporation (Seidman et al., 1997 or appendix 3N)

Alternate Protocol 2: Construction of CMV BAC Mutants by Two-Step Allele Replacement (Shuttle Mutagenesis)

 Materials
  • E. coli harboring the CMV BAC
  • Shuttle plasmid of the pST76 family (Posfai et al., 1997): e.g., pST76-KSR (Borst and Messerle, 2005)
  • LB medium (see recipe)
  • LB agar plates containing 17 µg/ml chloramphenicol and 25 µg/ml kanamycin (see recipe)
  • LB agar plates containing 17 µg/ml chloramphenicol (see recipe)
  • LB agar plates containing 17 µg/ml chloramphenicol plus 5% sucrose (see recipe), approximately 10 plates
  • LB agar plates containing 25 µg/ml kanamycin
  • 30°C thermomixer or shaking water bath
  • 30°C, 37°C, and 43°C incubators
  • Additional reagents and equipment for transforming bacteria by electroporation (Seidman et al., 1997 or appendix 3N), isolating DNA using a BAC miniprep procedure (see Basic Protocol), and analyzing DNA by restriction enzyme digestion (unit 10.8) and agarose gel electrophoresis (unit 10.4)

Support Protocol 1: Excision of the Selection Marker from Mutated CMV BACS by Flp Recombinase

 Materials
  • CMV BAC–containing E. coli strain (e.g., strain DH10B), typically recombination-incompetent (recA): obtain from researchers generating the CMV BAC of interest
  • Plasmid expressing Flp recombinase (e.g., pCP20; see Cherepanov and Wackernagel, 1995)
  • LB medium
  • LB agar plates containing 17 µg/ml chloramphenicol and 25 µg/ml ampicillin
  • LB agar plates containing 17 µg/ml chloramphenicol
  • LB agar plates containing 25 µg/ml kanamycin
  • Thermomixer
  • 30°C, 37°C, and 43°C incubators
  • Additional reagents and equipment for transforming bacterial cells by electroporation, isolating DNA using a BAC miniprep procedure (see Basic Protocol), and analyzing DNA by restriction enzyme digestion (unit 10.8).

Support Protocol 2: Insertion of Specific Sequences into a CMV BAC Using Flp-Mediated Recombination

 Materials
  • Selection marker flanked by suitable FRT sites (e.g., from an E. coli stock center)
  • E. coli strain (e.g., DH10B) containing the CMV BAC: obtain from researchers generating the CMV BAC of interest
  • Shuttle vector of the pOri6K-Kan series (see Borst and Messerle, 2005)
  • E. coli strain pir1 (Invitrogen)
  • LB with and without 17 µg/ml chloramphenicol plus 50 µg/ml ampicillin medium
  • LB agar plates containing 17 µg/ml chloramphenicol and 50 µg/ml ampicillin
  • LB agar plates containing 17 µg/ml chloramphenicol and 50 µg/ml kanamycin
  • 30°C shaking incubator
  • 30°C and 43°C incubators
  • Additional reagents and equipment for inserting a selection marker into a CMV BAC (Basic Protocol), excising a selection marker (Support Protocol 1, optional), constructing hybrid DNA molecules (Struhl, 1991), transforming bacteria by electroporation (Seidman et al., 1997 or appendix 3N), isolating DNA using a miniprep procedure (unit 10.3), and analyzing DNA using restriction analysis (unit 10.8)

Support Protocol 3: Reconstitution of CMV Mutants by Transfection into Permissive Cells

 Materials
  • 1 to 2 µg mCMV BAC DNA (see Basic Protocol)
  • Murine embryonic fibroblasts (MEF; see unit 19.7) or NIH/3T3 cells (ATCC# CRL-1658)
  • 6-well tissue culture plates
  • 5 × 105 human embryonic lung fibroblasts (MRC-5; ATCC# CCL-171)
  • TransFectin lipid reagent (Bio-Rad) or
  • SuperFect Transfection Reagent (Qiagen)
  • Complete medium (e.g., complete DMEM with 5% fetal calf serum; appendix 2A)
  • Additional reagents and equipment for growing eukaryotic cells in culture (see unit 10.15) and transfecting eukaryotic cells by electroporation (unit 10.15) or the calcium phosphate method (unit 10.13)

Support Protocol 4: Isolation of Viral DNA From Virus Particles of the CMV Mutant and Analysis of the Genomic Structure by Restriction Analysis and Southern Blotting

 Materials
  • Human foreskin fibroblasts (HFF; e.g., HFF-1; ATCC# SCRC-1041)
  • Complete medium (appendix 2A)
  • Supernatant from transfected cells and parental virus (Support Protocol 3), diluted to give an MOI of 0.1 (Support Protocol 5, steps 11 to 21)
  • 50 mM Tris×Cl pH 8.0 (appendix 2A)/ 1 mM MgCl2 / 100 µg/ml BSA
  • Benzonase (Sigma)
  • 0.5 M EDTA, pH 8.0 (appendix 2A)
  • 1% (w/v) SDS
  • 20 mg/ml proteinase K (Sigma)
  • Phenol/chloroform solution (see unit 10.1)
  • 35 mg/ml glycogen
  • 3 M sodium acetate, pH 5.2
  • Isopropanol
  • 70% ethanol
  • TE buffer
  • 10-cm tissue culture dishes
  • 37°C, 5% CO2 incubator
  • 15-ml centrifuge tubes
  • 15-ml ultracentrifuge tubes (Beckman)
  • Ultracentrifuge
  • 2-ml microcentrifuge tube
  • 56°C water bath or thermomixer
  • 200-ml pipet tips, with ends cut off
  • Additional reagents and equipment for analyzing DNA by restriction enzyme digestion (unit 10.8), agarose gel electrophoresis (unit 10.4), and Southern blot (unit 10.6A)

Support Protocol 5: Large Scale Preparation of hCMV Mutants

 Materials
  • Human foreskin fibroblasts (HFF; e.g., HFF-1; ATCC# SCRC-1041)
  • Supernatant from transfected MRC-5 cells (Support Protocol 4), diluted in complete medium to result an MOI of 0.1 (see steps 11 to 21 in this protocol)
  • Complete medium (e.g., see appendix 2A)
  • Skim milk solution (9.6 g skim milk powder in 100 ml sterile water; autoclave), optional
  • High-titer anti-hCMV patient serum (e.g., Sigma)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 100% methanol
  • Giemsa stain solution (e.g., Fluka), optional
  • 145-cm2 cell culture dishes
  • 37°C, 5% CO2 incubator
  • 500-ml and 250-ml centrifuge bottles, sterile
  • Refrigerated centrifuge and rotors (e.g., Heraeus Megafuge 1.0R or Beckmann J2-21 with rotors JA-10 and JA-14)
  • Dounce homogenizers with tight-fitting pestles
  • 15-ml centrifuge tubes
  • 0.5-ml microcentrifuge tubes, sterile
  • Inverse microscope
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Figures

  • Figure 10.32.1
    Mutagenesis of CMV BACs using linear DNA fragments. An antibiotic resistance gene (typically the kanamycin resistance gene, Knr) flanked by sequences homologous to the sequences directly upstream and downstream to the target gene (labeled with a star) in the CMV BAC is amplified by PCR. Then, the E. coli strain DH10B, carrying the plasmid pKD46, is transformed with the PCR product. The recombination enzymes expressed from pKD46 mediate homologous recombination between the linear PCR product and the CMV BAC. The target gene is replaced by the kanamycin resistance cassette. The scheme is not drawn to scale.

    View Image
  • Figure 10.32.2
    Schematic overview of the galK-based mutagenesis procedure. The galK cassette is amplified by PCR, using primers designed with overhangs such that 50 bp of the CMV sequences directly upstream and downstream of the intended integration site (labeled with the star) on the CMV BAC are incorporated into the PCR product. The resulting linear DNA fragment is transformed into E. coli SW102 bacteria expressing the bacteriophage genes red , , and , which mediate the recombination of the galK cassette into the CMV BAC. The now galK-positive bacteria are able to grow on minimal medium with galactose. In the second step the E. coli SW102 bacteria are transformed with a linear DNA fragment (typically an oligonucleotide) homologous to the CMV sequences directly upstream and downstream of the galK cassette, typically also containing the desired mutation (indicated by the circles). Following the replacement of the galK cassette by the second DNA fragment, the bacteria are plated on minimal medium agar plates containing 2-deoxy-d-galactose (DOG). The product of the galK gene, galactokinase, produces a toxic catabolite from DOG. For this reason, only colonies containing CMV BACs from which the galK cassette has been successfully removed can grow on these plates.

    View Image
  • Figure 10.32.3
    Two-step replacement method for mutating a CMV BAC. Bacteria containing the CMV BAC to be mutated are transformed with the shuttle plasmid harboring the mutant allele (indicated by the black rectangle) flanked by homologous sequences L (left side homology) and R (right side homology). In the first step, formation of co-integrates due to recombination via L or R leads to bacteria resistant to both chloramphenicol (Cmr) and kanamycin (Knr). Selection is done at 43°C to remove clones that contain the shuttle plasmid and the CMV BAC separately. In the second step, recombination via L or R results in the resolution of co-integrates to either the wild-type or the mutant CMV BAC. Streaking the bacteria on agar plates containing 5% sucrose allows the selection against clones still containing the co-integrate and the negative selection marker sacB, which prevents grow of bacteria in the presence of sucrose. The bacterial colonies that grow on the sucrose-containing plates harbor either a mutated or unchanged CMV BAC.

    View Image

Literature Cited

Literature Cited
    Borst, E.M. and Messerle, M. 2005. Analysis of human cytomegalovirus oriLyt sequence requirements in the context of the viral genome. J. Virol. 79:3615-3626.
    Borst, E.M., Hahn, G., Koszinowski, U.H., and Messerle, M. 1999. Cloning of the human cytomegalovirus (hCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: A new approach for construction of hCMV mutants. J. Virol. 73:8320-8329.
    Brune, W., Wagner, M., and Messerle, M. 2005. Manipulating cytomegalovirus genomes by BAC mutagenesis: Strategies and applications. In Cytomegaloviruses: Molecular Biology and Immunology. (M.J. Reddehase, ed.) pp. 63-89. Horizon Scientific Press, Hethersett, Norwich, UK.
    Chee, M.S., Bankier, A.T., Beck, S., Bohni, R., Brown, C.M., Cerny, R., Horsnell, T., Hutchison, C.A., Kouzarides, T., Martignetti, J.A., Preddie, E., Satchwell, S.C., Tomlinson, P., Weston, K.M., and Barrell, B.G. 1990. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr. Top. Microbiol. Immunol. 154:125-169.
    Cherepanov, P.P. and Wackernagel, W. 1995. Gene disruption in Escherichia coli: Tcr and Kmr cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158:9-14.
    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.
    Dolan, A., Cunningham, C., Hector, R.D., Hassan-Walker, A.F., Lee, L., Addison, C., Dargan, D.J., McGeoch, D.J., Gatherer, D., Emery, V.C., Griffiths, P.D., Sinzger, C., McSharry, B.P., Wilkinson, G.W., and Davison, A.J. 2004. Genetic content of wild-type human cytomegalovirus. J. Gen. Virol. 85:1301-1312.
    Dunn, W., Chou, C., Li, H., Hai, R., Patterson, D., Stolc, V., Zhu, H., and Liu, F. 2003. Functional profiling of a human cytomegalovirus genome. Proc. Natl. Acad. Sci. U.S.A. 100:14223-14228.
    Krmpotic, A., Bubic, I., Polic, B., Lucin, P., and Jonjic, S. 2003. Pathogenesis of murine cytomegalovirus infection. Microbes Infect. 5:1263-1277.
    Messerle, M., Crnkovic, I., Hammerschmidt, W., Ziegler, H., and Koszinowski, U.H. 1997. Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. U.S.A. 94:14759-14763.
    Murphy, E., Yu, D., Grimwood, J., Schmutz, J., Dickson, M., Jarvis, M.A., Hahn, G., Nelson, J.A., Myers, R.M., and Shenk, T.E. 2003. Coding potential of laboratory and clinical strains of human cytomegalovirus. Proc. Natl. Acad. Sci. U.S.A. 100:14976-14981.
    Posfai, G., Koob, M.D., Kirkpatrick, H.A., and Blattner, F.R. 1997. Versatile insertion plasmids for targeted genome manipulations in bacteria: Isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome. J. Bacteriol. 179:4426-4428.
    Rawlinson, W.D., Farrell, H.E., and Barrell, B.G. 1996. Analysis of the complete DNA sequence of murine cytomegalovirus. J. Virol. 70:8833-8849.
    Reddehase, M.J. 2005. Cytomegaloviruses: Molecular Biology and Immunology. Horizon Scientific Press, Hethersett, Norwich, UK.
    Reddehase, M.J., Simon, C.O., Podlech, J., and Holtappels, R. 2004. Stalemating a clever opportunist: Lessons from murine cytomegalovirus. Hum. Immunol. 65:446-455.
    Schlake, T. and Bode, J. 1994. Use of mutated Flp recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33:12746-12751.
    Seidman, C.E., Struhl, K., Sheen, J., and Jessen, T. 1997. Introduction of plasmid DNA into cells. Curr. Protoc. Mol. Biol. 37:1.8.1-1.8.10.
    Spaete, R.R. and Mocarski, E.S. 1987. Insertion and deletion mutagenesis of the human cytomegalovirus genome. Proc. Natl. Acad. Sci. U.S.A. 84:7213-7217.
    Struhl, K. 1991. Subcloning of DNA fragments. Curr. Protoc. Mol. Biol. 13:3.16.1-3.16.11.
    Tischer, B.K., von Einem, J., Kaufer, B., and Osterrieder, N. 2006. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40:191-197.
    Wagner, M., Jonjic, S., Koszinowski, U.H., and Messerle, M. 1999. Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution. J. Virol. 73:7056-7060.
    Warming, S., Costantino, N., Court, D.L., Jenkins, N.A., and Copeland, N.G. 2005. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33:e36.
    Yu, D., Ellis, H.M., Lee, E.C., Jenkins, N.A., Copeland, N.G., and Court, D.L. 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 97:5978-5983.
    Yu, D., Smith, G.A., Enquist, L.W., and Shenk, T. 2002. Construction of a self-excisable bacterial artificial chromosome containing the human cytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene. J. Virol. 76:2316-2328.
    Yu, D., Silva, M.C., and Shenk, T. 2003. Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc. Natl. Acad. Sci. U.S.A. 100:12396-12401.
    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.
 Key Reference
    Messerle et al., 1997. See above.

This paper describes the first successful cloning of a herpesvirus genome as a BAC and its manipulation with recombination techniques in E. coli.

 Internet Resources
    http://cgsc.biology.yale.edu

The E. coli Genetic Stock Center at Yale University can provide a number of useful plasmids and bacterial strains.

    http://recombineering.ncifcrf.gov

Dr. Court and colleagues at NCI Frederick provide important tools for recombineering. They also provide detailed protocols on their homepage as well as a FAQ section.

    http://www.genebridges.com

The company sells plasmids for Red/ET mutagenesis and offers a mutagenesis service.

     
 
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