Rapid, Efficient, and Modular Generation of Adenoviral Vectors via Isothermal Assembly

Yong Yang1, Yudan Chi1, Xinying Tang1, Hildegund C.J. Ertl2, Dongming Zhou1

1 Vaccine Research Center of Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, 2 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.26
DOI:  10.1002/0471142727.mb1626s113
Online Posting Date:  January, 2016
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Abstract

Adenoviral vectors have yielded promising results as carriers for gene transfer and vaccines in basic research and clinical applications. However, most common procedures to construct adenoviral vectors and manipulate adenovirus (Ad) genomes are complex and labor‐intensive. An easy and detailed protocol for the rapid, efficient, and modular generation of chimpanzee Ad serotype 68 (AdC68) as a molecular clone via isothermal assembly, which directionally assembles multiple DNA fragments in a single isothermal reaction without restriction enzymes or ligases, is presented. Any serotype of adenovirus with the sequence of genome known can be constructed as a molecular clone by this method. Recombinant adenoviral vectors can be created via one‐step isothermal assembly in <3 days, and recombinant Ads can be rescued within 8 days. This protocol is practical for manipulations of Ad genomes, because an entire Ad genome can be divided into specific fragments within modular plasmids. © 2016 by John Wiley & Sons, Inc.

Keywords: adenovirus genome; chimpanzee adenovirus 68 (AdC68); isothermal assembly; modular adenovirus molecular clones; recombinant adenovirus

     
 
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Table of Contents

  • Basic Protocol 1:  
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:  

  Materials
  • Chimpanzee Ad serotype 68 (AdC68) (also called SAdV‐25; ATCC #VR‐594, GenBank, no. AF394196.1)
  • DNeasy Blood & Tissue Kit (QIAGEN, cat. no. 69504) containing:
    • DNeasy mini spin columns in 2‐ml collections tubes
    • 2‐ml collection tubes
    • Buffer ATL
    • Buffer AL
    • Buffer AW1
    • Buffer AW2
    • Buffer AE
    • Proteinase K
  • Pronase (Roche, cat. no. 9036‐06‐0)
  • 96% to 100% ethanol
  • Milli‐Q autoclaved water
  • Primers (GenScript)
  • pNEB193 (New England Biolabs, cat. no. N3051S)
  • PrimeSTAR HS DNA polymerase, 5× PrimeSTAR buffer, deoxyribonucleotide triphosphate (dNTP) mixture (TaKaRa, cat. no. R010Q); Q5 High‐Fidelity DNA polymerase, 5× Q5 reaction buffer, dNTPs (New England Biolabs, cat. no. M0491S); and Phusion High‐Fidelity DNA polymerase (New England Biolabs, cat. no. M0530S)
  • DpnI (New England Biolabs, cat. no. R0176S)
  • QIAquick PCR Purification Kit (QIAGEN, cat. no. 28104)
  • Isothermal assembly mix (New England Biolabs)
  • 1× KCM buffer (see recipe)
  • Escherichia coli strain DH5α competent cells (Life Technologies, cat. no. 18265‐017)
  • Ampicillin‐containing LB plates (see recipe)
  • LB‐selective medium (see recipe)
  • QIAprep Spin Miniprep Kit (QIAGEN, cat. no. 27104)
  • PacI (New England Biolabs, cat. no. R01547S)
  • SbfI (New England Biolabs, cat. no. R0642S)
  • HpaI (New England Biolabs, cat. no. R0105V)
  • 0.8% and 1% agarose gels in TAE buffer
  • QIAEX II Gel Extraction Kit (QIAGEN, cat. no. 20021)
  • E. coli strain Stbl2 competent cells (Life Technologies, cat. no. 10268‐019)
  • BglII (New England Biolabs, cat. no. R0144V)
  • XhoI (New England Biolabs, cat. no. R0146V)
  • MfeI (New England Biolabs, cat. no. R0589S)
  • PcDNA6.2‐GW/EmGFP‐miR (Life Technologies, cat. no. K4936‐00)
  • NucleoBond Xtra Midi Plus (MACHEREY‐NAGEL, cat. no. 740412.50)
  • HEK 293 (ATCC # CCL‐243)
  • Dulbecco's modified Eagle's medium (DMEM; Life Technologies, cat. no. 11885‐084) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin
  • OPTI‐MEM (Life Technologies, cat. no. 31985‐088)
  • Lipofectamine 2000 transfection reagent (Life Technologies, cat. no. 11668‐019)
  • 1‐ml microcentrifuge tubes
  • Vortexer
  • 37°, 55°, 70°C water baths
  • Centrifuge
  • Thermal cycler
  • Nanodrop instrument
  • 30° and 37°C incubators with and without shaker
  • Gel apparatus and power supply
  • 1.5‐ml microcentrifuge tubes
  • 6‐well plates
  • 37°C, 5% CO 2 incubator
  • Fluorescence microscope
CAUTION: Wild‐type AdC68 is classified as a Biosafety Level 2. Accordingly, proper containment, sterile, and antiseptic measures should be used for all reagents, solutions, and equipment. Chlorine bleach should be used to disinfect the biohazard wastes containing Ads.CAUTION: GelRed nucleic acid gel stain is toxic. Wear gloves and dispose of waste according to appropriate guidelines.
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Figures

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Literature Cited

Literature Cited
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