Human Cytomegalovirus: Bacterial Artificial Chromosome (BAC) Cloning and Genetic Manipulation

Anne M. Paredes1, Dong Yu1

1 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14E.4
DOI:  10.1002/9780471729259.mc14e04s24
Online Posting Date:  February, 2012
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Abstract

The understanding of human cytomegalovirus (HCMV) biology was long hindered by the inability to perform efficient viral genetic analysis. This hurdle was recently overcome when the genomes of multiple HCMV strains were cloned as infectious bacterial artificial chromosomes (BACs). The BAC system takes advantage of the single‐copy F plasmid of E. coli that can stably carry large pieces of foreign DNA. In this system, a recombinant HCMV virus carrying a modified F plasmid is first generated in eukaryotic cells. Recombinant viral genomes are then isolated and recovered in E. coli as BAC clones. BAC‐captured viral genomes can be manipulated using prokaryotic genetics, and recombinant virus can be reconstituted from BAC transfection in eukaryotic cells. The BAC reverse genetic system provides a reliable and efficient method to introduce genetic alterations into the viral genome in E.coli and subsequently analyze their effects on virus biology in eukaryotic cells. Curr. Protoc. Microbiol. 24:14E.1.1‐14E.1.33. © 2012 by John Wiley & Sons, Inc.

Keywords: human cytomegalovirus; HCMV; betaherpesvirus; herpesvirus; bacterial artificial chromosome; BAC; mutagenesis; virus reconstitution

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of an HCMV Genome as a BAC Clone
  • Basic Protocol 2: Reconstitution of Infectious Virus from BAC Clones
  • Basic Protocol 3: Seamless Genetic Manipulation of the BAC‐Cloned Viral Genome by Linear Fragment‐Based Homologous Recombination (Linear Recombination)
  • Support Protocol 1: Generation of Electrocompetent, Recombination‐Ready E. coli for Linear Recombination
  • Basic Protocol 4: Rapid Tagging of a Viral ORF in an HCMV BAC Clone by FLP‐Mediated Site‐Specific Recombination
  • Basic Protocol 5: Develop a Protein Genetics–Based Inducible System to Manipulate Viral Protein Stability and Activity by Tagging Its ORF with a ddFKBP Tag
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of an HCMV Genome as a BAC Clone

  Materials
  • Primary human fibroblasts
  • Plasmid expressing HCMV protein pp71 (e.g., pYD‐C21; Baldick et al., ; Yu et al., )
  • Supplemented DMEM (see recipe)
  • 20% (w/v) D‐sorbitol solution in 50 mM Tris⋅Cl (pH 7.2), 1 mM MgCl 2, sterile
  • TE buffer (10 mM Tris⋅Cl, 0.1 mM EDTA, pH 8.0), sterile
  • 2× lysis buffer (see recipe)
  • Tris‐equilibrated phenol
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 70% and 100% ethanol, ice cold
  • BAC vector, such as pYD‐C223 derived from pYD‐C29 (Yu et al., )
  • Commercial electrocompetent E. coli DH10B cells (e.g., Invitrogen MegaX)
  • LB agar plates ( appendix 4A)
  • Commercial kit for purifying BAC DNA or low‐copy number plasmid DNA (e.g., Clontech Nucleobond Xtra Midi DNA purification kit)
  • PmeI
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • 3 M potassium acetate, pH 5.5 ( appendix 2A)
  • Agarose gel
  • Dulbecco's modified Eagle's medium with 4.5 g/liter of glucose (DMEM; e.g., D5796 from Sigma)
  • Dulbecco's modified phosphate‐buffered saline (DPBS, e.g., Hyclone, cat. no. SH30028.03)
  • Trypsin solution (0.05% trypsin, 0.005% penicillin, 0.01% streptomycin in DPBS), sterile
  • DMEM/agarose overlay solution, sterile (see recipe)
  • 1000× (2 mg/ml) puromycin in water, filter sterilized
  • Hirt solution I (10 mM Tris⋅Cl, 10 mM EDTA, pH 8.0), sterile
  • Proteinase K
  • 10% sodium dodecyl sulfate (SDS; appendix 2A), sterile
  • 5 M NaCl, sterile ( appendix 2A)
  • 10 mM Tris⋅Cl buffer, pH 8.0 ( appendix 2A), sterile
  • SOC medium (see recipe)
  • LB medium ( appendix 4A)
  • 1000× (15 mg/ml) chloramphenicol in ethanol, filter sterilized
  • Bacteria freezing solution (65% glycerol, 0.1 M MgSO 4, 25 mM Tris⋅Cl, pH 8.0), sterile
  • Miniprep solution I (50 mM Tris⋅Cl, 10 mM EDTA, pH 8.0; appendix 2A)
  • Miniprep solution II (200 mM NaOH, 1% SDS; appendix 2A)
  • Miniprep solution III (3.0 M potassium acetate buffer, pH 5.5; appendix 2A)
  • Isopropanol
  • 1 mg/ml RNase A ( appendix 2A)
  • 10‐ and 15‐cm tissue culture plates
  • 37°C, 5% CO 2 humidified incubator
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Refrigerated multipurpose centrifuge (e.g., Eppendorf model 5810R)
  • 1 × 3 1/2–in. ultra‐clear centrifuge tubes (Beckman)
  • 5‐ml serological pipets, sterile
  • Ultracentrifuge with SW28 swinging bucket rotor or equivalent (Beckman)
  • Wide‐orifice pipet tips, sterile
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • Spectrophotometer
  • 1‐, 2‐, and 4‐mm gap electroporation cuvettes
  • Electroporator (e.g., Bio‐Rad Gene Pulsar Xcell)
  • 6‐well tissue culture dishes
  • Light microscope
  • Inverted fluorescence tissue culture microscope
  • Cell scraper, sterile
  • Tube rotator
  • Additional reagents and equipment for PCR ( appendix 3D)

Basic Protocol 2: Reconstitution of Infectious Virus from BAC Clones

  Materials
  • Bacterial culture of viral BAC clone (see protocol 1)
  • Nucleobond Xtra Midi DNA purification kit (Clontech)
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 3 M potassium acetate, pH 5.5 ( appendix 2A)
  • 70% and 100% ethanol, ice cold and room temperature
  • EcoRI
  • 0.6% agarose gel
  • 15‐cm plates of nearly confluent primary human fibroblasts
  • Supplemented DMEM, prewarmed
  • HCMV BAC DNA
  • pp71‐expressing plasmid (e.g., pYD‐C21)
  • Plasmid expressing Cre recombinase (e.g., GS403; Smith and Enquist, )
  • Trypsin
  • Centrifuge
  • 1.5‐ml microcentrifuge tubes
  • Wide‐orifice pipet tips
  • Refrigerated microcentrifuge
  • Spectrophotometer
  • 10‐cm tissue culture plates
  • 4‐mm electroporation cuvette
  • Electroporator (e.g., BioRad Gene Pulsar)
  • 37°C cell culture incubator
  • Light microscope
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes

Basic Protocol 3: Seamless Genetic Manipulation of the BAC‐Cloned Viral Genome by Linear Fragment‐Based Homologous Recombination (Linear Recombination)

  Materials
  • Plasmid pYD‐C255 carrying Kan/galK dual selection cassette (Qian et al., )
  • 5′ Primer: 50‐bp viral homologous sequence + CCTGTTGACAATTAATCATCG
  • 3′ Primer: 50‐bp viral homologous sequence + CTCAGCAAAAGTTCGATTTA
  • Advantage HD polymerase (Clontech)
  • DpnI restriction enzyme
  • Agarose gel
  • Commercial kit for PCR purification or gel extraction (e.g., Invitrogen PureLink PCR purification kit or Quick Gel extraction kit)
  • Electroporation‐competent SW102 E. coli cells (Warming et al., ) containing HCMV BAC clone (see protocol 4)
  • SOC medium (see recipe)
  • LB plates containing 15 µg/ml chloramphenicol and 25 µg/ml kanamycin
  • LB plates with 40 µg/ml ampicillin
  • MacConkey indicator plates (see recipe)
  • DNA fragment with altered gene
  • 1× M9 solution (see recipe)
  • DOG‐negative selection plates (see recipe)
  • 37°C water bath shaker
  • Electroporator
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • 10‐ml culture tubes
  • Additional reagents and equipment for PCR ( appendix 3D) and BAC DNA minipreparations (see protocol 1)

Support Protocol 1: Generation of Electrocompetent, Recombination‐Ready E. coli for Linear Recombination

  Materials
  • SW102 E. coli cells (Warming et al., ) that contain the HCMV BAC clone grown on LB plates
  • LB medium ( appendix 4A)
  • Sterile water, ice cold
  • 10% (v/v) glycerol solution in water, sterile, ice cold
  • 15‐ml culture tubes
  • 1‐liter culture flasks
  • 42°C water bath shaker
  • 500‐ml flasks, sterile
  • Spectrophotometer
  • Refrigerated supercentrifuge and rotor
  • 50‐ml conical tubes
  • Refrigerated benchtop centrifuge

Basic Protocol 4: Rapid Tagging of a Viral ORF in an HCMV BAC Clone by FLP‐Mediated Site‐Specific Recombination

  Materials
  • Plasmid pYD‐C744 that carries the 3xFLAG‐FRT‐Kan/galK‐FRT cassette (Perng and Yu, unpub. observ.)
  • 5′ Primer: 50‐bp viral homology + (ATG)GACTACAAAGACCATGACGG
  • 3′ Primer: 50‐bp viral homology + (CTA)CGGGAAGTTCCTATTCTCTA
  • Advantage HD polymerase (Clontech)
  • SW105 E. coli cells that encode the arabinose‐inducible FLP recombinase (Warming et al., ) and carry the HCMV BAC clone
  • LB medium ( appendix 4A)
  • 10% (w/v) L (+)‐arabinose solution in water, sterile
  • LB plates with 15 µg/ml chloramphenicol
  • LB plates with 25 µg/ml kanamycin
  • Thermal cycler
  • Shaking incubator
  • Spectrophotometer
  • Additional reagents and equipment for PCR ( appendix 3D), linear recombination (see protocol 3), and miniprep of BAC DNA (see protocol 1)

Basic Protocol 5: Develop a Protein Genetics–Based Inducible System to Manipulate Viral Protein Stability and Activity by Tagging Its ORF with a ddFKBP Tag

  Materials
  • Plasmid pYD‐C630 that carries the cassette composed of the coding sequence of ddFKBP (110aa FKBP12 F36V L106P variant) (Banaszynski et al., ) followed by FRT‐Kan/galK‐FRT (Perng et al., )
  • 5′ Primer: 50‐bp viral homology sequence + ATGGGAGTGCAGGTGGAAACCATC
  • 3′ Primer: 50‐bp viral homology sequence + GCTGGAGCTCCACCGCGGGAAGTTC
  • Advantage HD polymerase (Clontech)
  • SW105 E. coli cells that encode the arabinose‐inducible FLP recombinase (Warming et al., ) and contain the HCMV BAC clone (see protocol 4)
  • 1000× (1 mM) Shield‐1 (Shld1; Cheminpharma) solution in ethanol, stored at −20°C
  • Anti‐FKBP12 mouse monoclonal antibody (BD Bioscience, cat. no. 610808)
  • 1 × 3 1/2–in. ultra‐clear centrifuge tubes
  • 5‐ml pipets
  • Centrifuge with a SW28 swinging bucket rotor (or equivalent)
  • Additional reagents and equipment for PCR ( appendix 3D), linear recombination (see protocol 3), miniprep BAC DNA (see protocol 1), arabinose induction and FLP/FRT recombination (see protocol 5)
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Figures

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Literature Cited

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