Production of Papillomavirus‐Based Gene Transfer Vectors

Christopher B. Buck1, Cynthia D. Thompson1

1 National Cancer Institute, Center for Cancer Research, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.1
DOI:  10.1002/0471143030.cb2601s37
Online Posting Date:  December, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Papillomaviruses are a diverse group of pathogens that infect the skin and mucosal tissues of humans and various animal species. The viral genome is a circular, double‐stranded DNA molecule ∼8‐kb in length. The non‐enveloped papillomavirus capsid is composed of a virally encoded major coat protein, L1, and a minor coat protein, L2. L1 and L2 co‐assemble when expressed in mammalian cells, and can promiscuously encapsidate essentially any <8‐kb plasmid present in the cell nucleus. In the last several years, there has been rapid development of techniques for intracellular production of papillomavirus‐based gene transfer vectors (also known as pseudoviruses). This unit outlines the production and propagative amplification of papillomaviral vectors. The system represents a highly tractable method for converting pre‐existing mammalian expression plasmids into infectious virus stocks. The resulting vectors have utility for in vitro, as well as in vivo gene delivery applications. Curr. Protoc. Cell Biol. 37:26.1.1‐26.1.19. © 2007 by John Wiley & Sons, Inc.

Keywords: papillomavirus; HPV; pseudovirus; pseudovirion; virus‐like particle; VLP

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of a Papillomaviral Vector Stock by Transfection
  • Basic Protocol 2: Propagative Amplification of a Papillomaviral Vector Stock
  • Alternate Protocol 1: Production of Amplification‐Incompetent Vector Stocks
  • Alternate Protocol 2: Bulk Production of Papillomavirus Capsids
  • Support Protocol 1: Purification of Vector Stocks Using Optiprep Gradients
  • Support Protocol 2: Purification of Vector Stocks Using Agarose Gel Filtration
  • Support Protocol 3: Screening Fractions for the Presence of Capsids
  • Support Protocol 4: Concentration of Vector Stocks
  • Support Protocol 5: Titering the Infectivity of Papillomaviral Vector Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Generation of a Papillomaviral Vector Stock by Transfection

  Materials
  • DMEM (Dulbecco's modified Eagle medium ) with 10% (v/v) FBS (DMEM‐10)
  • Fetal bovine serum, heat‐inactivated at 56°C for 30 min (FBS)
  • 293TT cells
  • 0.05% (w/v) trypsin/EDTA solution (Invitrogen or other supplier)
  • 2× freeze medium: 82% (v/v) FBS/18% (v/v) dimethylsulfoxide
  • 50 mg/ml hygromycin B stock (Roche)
  • Lipofectamine 2000 (Invitrogen)
  • OptiMEM‐I (Invitrogen)
  • p16L1L2 plasmid
  • <8‐kb mammalian expression plasmid with SV40 ori (see )
  • DPBS‐Mg (see recipe)
  • 10% (v/v) Brij‐58 (see recipe)
  • RNase A/T1 cocktail (Ambion, no. AM2286) or RNase A stock
  • 225‐cm2 and 75‐cm2 cell culture flask
  • Humidified 37°C, 5% CO 2 incubator
  • 37°C water bath
  • 2‐ml gasketed cryovials
  • Cryocooler (e.g., Nalgene Mr. Frosty isopropanol bath) chilled to 4°C
  • Liquid nitrogen freezer
  • 15‐ml conical centrifuge tubes
  • 1.5‐ml siliconized centrifuge tubes, preferably screw‐cap (e.g., VWR, no, 60828‐818)
  • Siliconized pipet tips (optional)

Basic Protocol 2: Propagative Amplification of a Papillomaviral Vector Stock

  Materials
  • 293TT cells
  • DMEM with 10% (v/v) FBS
  • Vector seed stock ( protocol 1)
  • 225‐cm2 flask
  • Humidified 37°C 5% CO 2 incubator

Alternate Protocol 1: Production of Amplification‐Incompetent Vector Stocks

  • Benzonase endonuclease (∼250 U/µl; Sigma‐Aldrich or other supplier)
  • Plasmid Safe ATP‐Dependent DNase (10 U/µl; Epicentre)
  • 5 M NaCl
  • Additional reagents and equipment for production of an initial vector seed stock ( protocol 1)

Alternate Protocol 2: Bulk Production of Papillomavirus Capsids

  • 46% (v/v) Optiprep/DPBS (see recipe)
  • DPBS/0.8 M NaCl (prepare by adding 0.15 volume of 5 M NaCl to DPBS)
  • Thin‐wall 1/2 × 2–in. Polyallomer tubes (Beckman, no. 326819) or appropriate ultracentrifuge tubes
  • 3‐ml syringe fitted with a 2‐in. or longer large‐bore (∼16‐G) needle
  • Parafilm
  • Ultracentrifuge
  • Beckman SW‐55 ultracentrifuge rotor (or other swing‐bucket rotor)
  • Ring stand with tube clamp
  • Siliconized collection tubes
  • ∼25‐G needle

Support Protocol 1: Purification of Vector Stocks Using Optiprep Gradients

  • DPBS‐BSA (see recipe)
  • DPBS/0.5 M NaCl (prepare by adding 0.075 volume of 5 M NaCl to DPBS)
  • Benzonase nuclease (Sigma; optional)
  • 0.05% (w/v) NaN 3 or other preservative
  • Vacuum source (optional)
  • Bell jar or side‐arm vacuum flask for degassing solutions (optional)
  • Ring stand with clamp
  • 5 ml gravity‐flow columns with caps and frits (Pierce or other supplier)
  • 1‐ or 5‐ml pipet
  • 2% (w/v) agarose beads (50‐ to 150‐µm diameter; Agarose Bead Technologies, no. A‐1020‐S) or 1.4% agarose beads (Bioscience Beads)
  • Parafilm

Support Protocol 2: Purification of Vector Stocks Using Agarose Gel Filtration

  • Electrophoresis‐grade agarose
  • Tris‐acetate‐EDTA (TAE) electrophoresis buffer (or other agarose gel electrophoresis buffer, see appendix 2A)
  • Phosphate‐buffered saline ( appendix 2A), optional
  • Proteinase K (e.g., Qiagen, no. 19131)
  • 0.5 M EDTA
  • 10% SDS
  • DNA loading dye (Voytas, )
  • DNA marker ladder (kilobase‐range; Invitrogen or other supplier)
  • 1 to 10 ng sample of supercoiled p16L1L2 and/or 1 to 10 ng of the expression plasmid of interest
  • SYBR Green‐I (Invitrogen/Molecular Probes or other supplier)
  • UV (or blue light) gel documentation imaging device
  • 16‐ × 16–cm tray
  • 12‐well combs
  • Agarose gel electrophoresis apparatus (see appendix 3A)

Support Protocol 3: Screening Fractions for the Presence of Capsids

  Materials
  • Cells, e.g., 293TT
  • DMEM‐10
  • Vector stocks to be tested
  • Dulbecco's phosphate‐buffered saline (DPBS) supplemented with 1% (v/v) FBS
  • ι‐carrageenan (e.g., Sigma, no. C4014)
  • 24‐well plate
  • 2.5‐µl pipettor
  • 1000‐µl pipettor
  • Fluorescent microscope
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Buck, C.B., Pastrana, D.V., Lowy, D.R., and Schiller, J.T. 2004. Efficient intracellular assembly of papillomaviral vectors. J. Virol. 78:751‐757.
   Buck, C.B., Thompson, C.D., Pang, Y.Y., Lowy, D.R., and Schiller, J.T. 2005. Maturation of papillomavirus capsids. J. Virol. 79:2839‐2846.
   Buck, C.B., Thompson, C.D., Roberts, J.N., Muller, M., Lowy, D.R., and Schiller, J.T. 2006. Carrageenan is a potent inhibitor of papillomavirus infection. PLoS Pathog. 2:e69.
   Buck, C.B., Cheng, N., Thompson, C.D., Lowy, D.R., Steven, A.C., Schiller, J.T., and Trus, B.L. Arrangement of L2 within the papillomavirus capsid. Submitted for publication.
   Choi, V.W., Asokan, A., Haberman, R.D., and Samulski, R.J. 2007. Production of recombinant adeno‐associated viral vectors for in vitro and in vivo use. Curr. Protoc. Mol. Biol. 78:16.25.1‐16.25.24.
   Cooper, M.J., Lippa, M., Payne, J.M., Hatzivassiliou, G., Reifenberg, E., Fayazi, B., Perales, J.C., Morrison, L.J., Templeton, D., Piekarz, R.L., et al. 1997. Safety‐modified episomal vectors for human gene therapy. Proc. Natl. Acad. Sci. U.S.A. 94:6450‐6455.
   Fu, X.Y. and Manley, J.L. 1987. Factors influencing alternative splice site utilization in vivo. Mol. Cell Biol. 7:738‐748.
   Leder, C., Kleinschmidt, J.A., Wiethe, C., and Muller, M. 2001. Enhancement of capsid gene expression: preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes. J. Virol. 75:9201‐9209.
   Okuley, S., Call, M., Mitchell, T., Hu, B., and Woodworth, M.E. 2003. Relationship among location of T‐antigen‐induced DNA distortion, auxiliary sequences, and DNA replication efficiency. J. Virol. 77:10651‐10657.
   Roberts, J.N., Buck, C.B., Thompson, C.D., Kines, R., Bernardo, M., Choyke, P.L., Lowy, D.R., and Schiller, J.T. 2007. Genital transmission of HPV in a mouse model is potentiated by nonoxynol‐9 and inhibited by carrageenan. Nat. Med. (in press).
   Roden, R.B., Lowy, D.R., and Schiller, J.T. 1997. Papillomavirus is resistant to desiccation. J. Infect. Dis. 176:1076‐1079.
   Shi, L., Sanyal, G., Ni, A., Luo, Z., Doshna, S., Wang, B., Graham, T.L., Wang, N., and Volkin, D.B. 2005. Stabilization of human papillomavirus virus‐like particles by non‐ionic surfactants. J. Pharm. Sci. 94:1538‐1551.
   Smith, R.W., Steffen, C., Grosse, F., and Nasheuer, H.P. 2002. Species specificity of simian virus 40 DNA replication in vitro requires multiple functions of human DNA polymerase alpha. J. Biol. Chem. 277:20541‐20548.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
Internet Resources
  http://home.ccr.cancer.gov/LCO/
  The Laboratory of Cellular Oncology maintains this website listing plasmid maps and pseudovirus‐related technical protocols.
  http://www.axis‐shield.com/densityhome/density/dapp.htm
  Axis‐Shield offers a useful handbook containing information about the use of Optiprep.
  http://www.invitrogen.com/content/sfs/productnotes/F_051025_MammalianExpressionVectors‐TS‐TL‐MKT‐HL.pdf
  Invitrogen offers a brochure comparing various commonly‐used mammalian promoters.
  http://www.cdc.gov/od/ohs/pdffiles/4th%20BMBL.pdf
  The Centers for Disease Control and Prevention's handbook entitled “Biosafety in Microbiological and Biomedical Laboratories” discusses procedures for the handling of biohazardous substances.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library