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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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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Basic Protocol 1:  

  • 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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Literature Cited

Literature Cited
  Breyer, B., Jiang, W., Cheng, H., Zhou, L., Paul, R., Feng, T., and He, T.C. 2001. Adenoviral vector‐mediated gene transfer for human gene therapy. Curr. Gene Ther. 1:149‐162. doi: 10.2174/1566523013348689.
  Carlisle, R., Choi, J., Bazan‐Peregrino, M., Laga, R., Subr, V., Kostka, L., Ulbrich, K., Coussios, C.C., and Seymour, L.W. 2013. Enhanced tumor uptake and penetration of virotherapy using polymer stealthing and focused ultrasound. J. Natl. Cancer Inst. 105:1701‐1710. doi: 10.1093/jnci/djt305.
  Dormitzer, P.R., Suphaphiphat, P., Gibson, D.G., Wentworth, D.E., Stockwell, T.B., Algire, M.A., Alperovich, N., Barro, M., Brown, D.M., Craig, S., Dattilo, B.M., Denisova, E.A., De Souza, I., Eickmann, M., Dugan, V.G., Ferrari, A., Gomila, R.C., Han, L., Judge, C., Mane, S., Matrosovich, M., Merryman, C., Palladino, G., Palmer, G.A., Spencer, T., Strecker, T., Trusheim, H., Uhlendorff, J., Wen, Y., Yee, A.C., Zaveri, J., Zhou, B., Becker, S., Donabedian, A., Mason, P.W., Glass, J.I., Rappuoli, R., and Venter, J.C. 2013. Synthetic generation of influenza vaccine viruses for rapid response to pandemics. Sci. Transl. Med. 5:185ra168. doi: 10.1126/scitranslmed.3006368.
  Ewer, K.J., O'Hara, G.A., Duncan, C.J., Collins, K.A., Sheehy, S.H., Reyes‐Sandoval, A., Goodman, A.L., Edwards, N.J., Elias, S.C., Halstead, F.D., Longley, R.J., Rowland, R., Poulton, I.D., Draper, S.J., Blagborough, A.M., Berrie, E., Moyle, S., Williams, N., Siani, L., Folgori, A., Colloca, S., Sinden, R.E., Lawrie, A.M., Cortese, R., Gilbert, S.C., Nicosia, A., and Hill, A.V. 2013. Protective CD8 +T‐cell immunity to human malaria induced by chimpanzee adenovirus‐MVA immunisation. Nat. Commun. 4:2836. doi: 10.1038/ncomms3836.
  Gibson, D.G. 2011. Enzymatic assembly of overlapping DNA fragments. Meth. Enzymol. 498:349‐361. doi: 10.1016/B978-0-12-385120-8.00015-2.
  Gibson, D.G., Smith, H.O., Hutchison, C.A., 3rd, Venter, J.C., and Merryman, C. 2010. Chemical synthesis of the mouse mitochondrial genome. Nat. Methods 7:901‐903. doi: 10.1038/nmeth.1515.
  Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., 3rd, and Smith, H.O. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6:343‐345. doi: 10.1038/nmeth.1318.
  Graham, F.L. and Prevec, L. 1995. Methods for construction of adenovirus vectors. Mol. Biotechnol. 3:207‐220. doi: 10.1007/BF02789331.
  Guye, P., Li, Y., Wroblewska, L., Duportet, X., and Weiss, R. 2013. Rapid, modular and reliable construction of complex mammalian gene circuits. Nucleic Acids Res. 41:e156. doi: 10.1093/nar/gkt605.
  Heilbronn, R. and Weger, S. 2010. Viral vectors for gene transfer: Current status of gene therapeutics. Handb. Exp. Pharmacol. 143‐170. doi: 10.1007/978-3-642-00477-3_5.
  Janssen, J.M., Liu, J., Skokan, J., Goncalves, M.A., and de Vries, A.A. 2013. Development of an AdEasy‐based system to produce first‐ and second‐generation adenoviral vectors with tropism for CAR‐ or CD46‐positive cells. J. Gene Med. 15:1‐11. doi: 10.1002/jgm.2687.
  Kaufmann, J.K. and Nettelbeck, D.M. 2012. Virus chimeras for gene therapy, vaccination, and oncolysis: Adenoviruses and beyond. Trends Mol. Med. 18:365‐376. doi: 10.1016/j.molmed.2012.04.008.
  Lasaro, M.O. and Ertl, H.C. 2009. New insights on adenovirus as vaccine vectors. Mol. Ther. 17:1333‐1339. doi: 10.1038/mt.2009.130.
  Le, C.T., Gray, G.C., and Poddar, S.K. 2001. A modified rapid method of nucleic acid isolation from suspension of matured virus: Applied in restriction analysis of DNA from an adenovirus prototype strain and a patient isolate. J. Med. Microbiol. 50:571‐574. doi: 10.1099/0022-1317-50-6-571.
  Legrand, V., Leissner, P., Winter, A., Mehtali, M., and Lusky, M. 2002. Transductional targeting with recombinant adenovirus vectors. Curr. Gene Ther. 2:323‐339. doi: 10.2174/1566523023347823.
  Lie, A.L.M., Bakker, C.T., Wesseling, J.G., and Bosma, P.J. 2005. AdEasy‐based cloning system to generate tropism expanded replicating adenoviruses expressing transgenes late in the viral life cycle. Gene Ther. 12:1347‐1352. doi: 10.1038/sj.gt.3302546.
  Liu, J., O'Brien, K.L., Lynch, D.M., Simmons, N.L., La Porte, A., Riggs, A.M., Abbink, P., Coffey, R.T., Grandpre, L.E., Seaman, M.S., Landucci, G., Forthal, D.N., Montefiori, D.C., Carville, A., Mansfield, K.G., Havenga, M.J., Pau, M.G., Goudsmit, J., and Barouch, D.H. 2009. Immune control of an SIV challenge by a T‐cell‐based vaccine in Rhesus monkeys. Nature 457:87‐91. doi: 10.1038/nature07469.
  Luo, J., Deng, Z.L., Luo, X., Tang, N., Song, W.X., Chen, J., Sharff, K.A., Luu, H.H., Haydon, R.C., Kinzler, K.W., Vogelstein, B., and He, T.C. 2007. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat. Protoc. 2:1236‐1247. doi: 10.1038/nprot.2007.135.
  Mitchell, L.A., Cai, Y., Taylor, M., Noronha, A.M., Chuang, J., Dai, L., and Boeke, J.D. 2013. Multichange isothermal mutagenesis: A new strategy for multiple site‐directed mutations in plasmid DNA. ACS Synth. Biol. 2:473‐477. doi: 10.1021/sb300131w.
  Mizuguchi, H. and Kay, M.A. 1998. Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method. Hum. Gene Ther. 9:2577‐2583. doi: 10.1089/hum.1998.9.17-2577.
  Murakami, P., Pungor, E., Files, J., Do, L., van Rijnsoever, R., Vogels, R., Bout, A., and McCaman, M. 2002. A single short stretch of homology between adenoviral vector and packaging cell line can give rise to cytopathic effect‐inducing, helper‐dependent E1‐positive particles. Hum. Gene Ther. 13:909‐920. doi: 10.1089/10430340252939023.
  Murakami, M., Ugai, H., Belousova, N., Pereboev, A., Dent, P., Fisher, P.B., Everts, M., and Curiel, D.T. 2010. Chimeric adenoviral vectors incorporating a fiber of human adenovirus 3 efficiently mediate gene transfer into prostate cancer cells. Prostate 70:362‐376.
  Ng, P., Parks, R.J., Cummings, D.T., Evelegh, C.M., Sankar, U., and Graham, F.L. 1999. A high‐efficiency Cre/loxP‐based system for construction of adenoviral vectors. Hum. Gene Ther. 10:2667‐2672. doi: 10.1089/10430349950016708.
  Peruzzi, D., Dharmapuri, S., Cirillo, A., Bruni, B.E., Nicosia, A., Cortese, R., Colloca, S., Ciliberto, G., La Monica, N., and Aurisicchio, L. 2009. A novel chimpanzee serotype‐based adenoviral vector as delivery tool for cancer vaccines. Vaccine 27:1293‐1300. doi: 10.1016/j.vaccine.2008.12.051.
  Saxena, M., Van, T.T., Baird, F.J., Coloe, P.J., and Smooker, P.M. 2013. Pre‐existing immunity against vaccine vectors—Friend or foe? Microbiology 159:1‐11. doi: 10.1099/mic.0.049601-0.
  Schaack, J., Langer, S., and Guo, X. 1995. Efficient selection of recombinant adenoviruses by vectors that express beta‐galactosidase. J. Virol. 69:3920‐3923.
  Seregin, S.S. and Amalfitano, A. 2009. Overcoming pre‐existing adenovirus immunity by genetic engineering of adenovirus‐based vectors. Expert Opin. Biol. Ther. 9:1521‐1531. doi: 10.1517/14712590903307388.
  Seshidhar Reddy, P., Ganesh, S., Limbach, M.P., Brann, T., Pinkstaff, A., Kaloss, M., Kaleko, M., and Connelly, S. 2003. Development of adenovirus serotype 35 as a gene transfer vector. Virology 311:384‐393. doi: 10.1016/S0042-6822(03)00161-2.
  Singarapu, K., Pal, I., and Ramsey, J.D. 2013. Polyethylene glycol‐grafted polyethylenimine used to enhance adenovirus gene delivery. J. Biomed. Mater. Res. A 101:1857‐1864. doi: 10.1002/jbm.a.34483.
  Stone, D., Ni, S., Li, Z.Y., Gaggar, A., DiPaolo, N., Feng, Q., Sandig, V., and Lieber, A. 2005. Development and assessment of human adenovirus type 11 as a gene transfer vector. J. Virol. 79:5090‐5104. doi: 10.1128/JVI.79.8.5090-5104.2005.
  Tan, R., Li, C., Jiang, S., and Ma, L. 2006. A novel and simple method for construction of recombinant adenoviruses. Nucleic Acids Res. 34:e89. doi: 10.1093/nar/gkl449.
  Torella, J.P., Boehm, C.R., Lienert, F., Chen, J.H., Way, J.C., and Silver, P.A. 2014. Rapid construction of insulated genetic circuits via synthetic sequence‐guided isothermal assembly. Nucleic Acids Res. 42:681‐689. doi: 10.1093/nar/gkt860.
  Xiang, Z., Gao, G., Reyes‐Sandoval, A., Cohen, C.J., Li, Y., Bergelson, J.M., Wilson, J.M., and Ertl, H.C. 2002. Novel, chimpanzee serotype 68‐based adenoviral vaccine carrier for induction of antibodies to a transgene product. J. Virol. 76:2667‐2675. doi: 10.1128/JVI.76.6.2667-2675.2002.
  Xiang, W., Man, X., Chao, Z., Yong, Y., Yudan, C., Xinying, T., Hongbo, Z., Sidong, X., Luogang, Y., and Dongming, Z. 2014. Neutralizing antibody responses to enterovirus and adenovirus in healthy adults in China. Emerging Microb. Infect. 3:e30. doi: 10.1038/emi.2014.30.
  Xu, Z.L., Mizuguchi, H., Sakurai, F., Koizumi, N., Hosono, T., Kawabata, K., Watanabe, Y., Yamaguchi, T., and Hayakawa, T. 2005. Approaches to improving the kinetics of adenovirus‐delivered genes and gene products. Adv. Drug Deliv. Rev. 57:781‐802. doi: 10.1016/j.addr.2004.12.010.
  Youil, R., Toner, T.J., Su, Q., and Kaslow, D.C. 2001. Rapid method for the isolation of full length adenoviral genomes by bacterial intermolecular homologous recombination. J. Virol. Methods 92:91‐97. doi: 10.1016/S0166-0934(00)00280-9.
  Yu, D., Jin, C., Ramachandran, M., Xu, J., Nilsson, B., Korsgren, O., Le Blanc, K., Uhrbom, L., Forsberg‐Nilsson, K., Westermark, B., Adamson, R., Maitland, N., Fan, X., and Essand, M. 2013. Adenovirus serotype 5 vectors with Tat‐PTD modified hexon and serotype 35 fiber show greatly enhanced transduction capacity of primary cell cultures. PloS One 8:e54952. doi: 10.1371/journal.pone.0054952.
  Zahn, R., Gillisen, G., Roos, A., Koning, M., van der Helm, E., Spek, D., Weijtens, M., Grazia Pau, M., Radosevic, K., Weverling, G.J., Custers, J., Vellinga, J., Schuitemaker, H., Goudsmit, J., and Rodriguez, A. 2012. Ad35 and ad26 vaccine vectors induce potent and cross‐reactive antibody and T‐cell responses to multiple filovirus species. PloS One 7:e44115. doi: 10.1371/journal.pone.0044115.
  Zaiss, A.K., Machado, H.B., and Herschman, H.R. 2009. The influence of innate and pre‐existing immunity on adenovirus therapy. J. Cell. Biochem. 108:778‐790. doi: 10.1002/jcb.22328.
  Zak, D.E., Andersen‐Nissen, E., Peterson, E.R., Sato, A., Hamilton, M.K., Borgerding, J., Krishnamurty, A.T., Chang, J.T., Adams, D.J., Hensley, T.R., Salter, A.I., Morgan, C.A., Duerr, A.C., De Rosa, S.C., Aderem, A., and McElrath, M.J. 2012. Merck Ad5/HIV induces broad innate immune activation that predicts CD8(+) T‐cell responses but is attenuated by preexisting Ad5 immunity. Proc. Natl. Acad. Sci. U.S.A. 109:E3503‐3512. doi: 10.1073/pnas.1208972109.
  Zeng, M., Smith, S.K., Siegel, F., Shi, Z., Van Kampen, K.R., Elmets, C.A., and Tang, D.C. 2001. AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination. BioTechniques 31:260‐262.
  Zhang, J., Tarbet, E.B., Toro, H., and Tang, D.C. 2011. Adenovirus‐vectored drug‐vaccine duo as a potential driver for conferring mass protection against infectious diseases. Expert Rev. Vaccines 10:1539‐1552. doi: 10.1586/erv.11.141.
  Zhou, D., Zhou, X., Bian, A., Li, H., Chen, H., Small, J.C., Li, Y., Giles‐Davis, W., Xiang, Z., and Ertl, H.C. 2010. An efficient method of directly cloning chimpanzee adenovirus as a vaccine vector. Nat. Protoc. 5:1775‐1785. doi: 10.1038/nprot.2010.134.
  Zhu, Y.M., Yu, Z., Cai, H., Gao, Y.R., Dong, X.M., Li, Z.L., Shi, H.F., Meng, Q.F., Lu, C., and Xue, F. 2011. Isolation, identification, and complete genome sequence of a bovine adenovirus type 3 from cattle in China. Virol. J. 8:557. doi: 10.1186/1743-422X-8-557.
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