Production and Titration of Lentiviral Vectors

Patrick Salmon1, Didier Trono2

1 Department of Neuroscience, Faculty of Medicine, University of Geneva, Geneva, 2 School of Life Sciences, École Polytechnique Fédérale de, Lausanne and “Frontiers in Genetics,” National Center for Competence in Research, Lausanne
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.10
DOI:  10.1002/0471142905.hg1210s54
Online Posting Date:  July, 2007
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Abstract

Lentiviral vectors have emerged over the last decade as powerful, reliable and safe tools for stable gene transfer in a wide variety of mammalian cells. Unlike other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells. This is why LVs are becoming useful and promising tools for future gene and cell therapy approaches. Lentivectors (LVs) derived from HIV‐1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility. These latest designs are reviewed in this unit. This unit also describes protocols for production and titration of LVs that can be implemented in a research laboratory setting, with an emphasis on standardization to improve transposability of results between laboratories Curr. Protoc. Hum. Genet. 54:12.10.1‐12.10.24. © 2007 by John Wiley & Sons, Inc.

Keywords: Lentiviral vectors; gene therapy; gene delivery

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of High‐Titer HIV‐1‐Based Vector Stocks by Transient Transfection of 293T Cells
  • Support Protocol 1: Determination of Total Vector Concentration Using Anti‐p24 Immunoassay
  • Support Protocol 2: Biological Titration of Lentivectors Using Flow Cytometry
  • Support Protocol 3: Biological Titration of Lentivectors by Quantitative PCR (QPCR)
  • Support Protocol 4: RCR Assay for LVS: Real‐Time Quantitative PCR (QPCR) Detection of Replication‐Competent Recombinants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Production of High‐Titer HIV‐1‐Based Vector Stocks by Transient Transfection of 293T Cells

  Materials
  • 293T/17 cells (ATCC cat. no. SD‐3515)
  • Dulbecco's modified Eagle medium/10% FBS (DMEM‐10; appendix 2D)
  • 0.05% trypsin/EDTA (e.g., Invitrogen)
  • Plasmids (available from the Trono lab, http://tronolab.epfl.ch):
    • pMD2G (encoding the VSV G envelope protein)
    • pWPT‐GFP (second‐generation transfer vector, abbreviated “pWPT”)
    • pRRL‐cPPT‐PGK‐GFP‐W‐SIN (third‐generation transfer vector, abbreviated “pRRL”)
    • psPAX2 (encoding HIV‐1 Gag, Pol, Tat and Rev proteins)
    • pMDLgag/polRRE (encoding the HIV‐1 Gag and Pol proteins)
    • pRSVrev (encoding the HIV‐1 Rev protein)
  • TE buffer, pH 8.0 ( appendix 2A)
  • Buffered water (see recipe)
  • 0.5 M CaCl 2 (see recipe)
  • 2× HeBS (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2D), 37°C
  • 75% (v/v) ethanol in spray bottle
  • 20% (w/v) sucrose (SigmaUltra from Sigma), filter‐sterilized through 0.22‐µm filter (store at 4°C)
  • CellGro Stem Cell Growth Medium (CellGenix GmbH, http://www.cellgenix.com/; optional, if subsequent experiments require absence of serum)
  • Phosphate‐buffered saline containing Ca2+ and Mg2+ (e.g., Invitrogen; optional, if subsequent experiments require absence of protein)
  • 10‐cm tissue culture dishes
  • 15‐ and 50‐ml conical centrifuge tubes, sterile
  • 50‐ml syringes and 0.45‐µm pore size PVDF filters
  • 30‐ml Beckman Konical ultracentrifuge tubes (Beckman Coulter)
  • Ultracentrifuge with SW 28 rotor (Beckman Coulter) or equivalent
  • Additional reagents and equipment for tissue culture ( appendix 3G) and quantitation of DNA by absorption spectroscopy ( appendix 3D)
CAUTION: P2 practices require that open tubes always be handled in the laminar flow hood. Tubes can be taken out of the laminar flow only when they are closed, and that they be sprayed with 75% ethanol. All solid waste and plasticware must be discarded in a trash bin in the laminar flow hoods and all liquids must be aspirated into a liquid waste bottle containing fresh concentrated bleach. Refill the liquid waste bottle with fresh bleach when the color of the liquid is no longer yellow. When full, bags are closed inside the laminar flow hood, then autoclaved. When full, and at least 15 min after neutralization with fresh bleach, the liquid waste bottle can be emptied into a regular sink. In case of a major spill of vector‐containing liquid, absorb liquid with paper towels and neutralize with fresh concentrated bleach prior to disposal. In case there is a leak in the SW 28 buckets, remove the tubes in the hood, fill the buckets with 75% ethanol, and invert them several times. Leave under the hood for ≥20 min. Discard the 75% ethanol and remove the conical adapters under the hood. Spray the adapters with 75% ethanol and leave them under the hood for >20 min.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified, 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Determination of Total Vector Concentration Using Anti‐p24 Immunoassay

  Materials
  • Lentiviral vector sample for titration (see protocol 1) and positive control (provided with p24 ELISA kit)
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • 5% (v/v) Triton X‐100 (store at room temperature)
  • p24 ELISA kit (Perkin‐Elmer cat. no. NEK05000 1KT) including:
    • Anti‐p24‐coated ELISA 96‐well plates (or well strips)
    • Adhesive plate covers
    • p24 wash buffer
    • Biotinylated anti‐p24 polyclonal antibody for primary reaction
    • Streptavidin–horseradish peroxidase (HRP) for secondary reaction
    • Substrate: o‐phenylenediamine HCl (OPD) tablets
    • Substrate diluent
    • Stop solution: 4 N sulfuric acid
  • ELISA plate reader with 492 nm filter

Support Protocol 2: Biological Titration of Lentivectors Using Flow Cytometry

  Materials
  • HeLa cells (ATCC cat. no. CCL‐2)
  • Dulbecco's modified Eagle medium/10% FBS (DMEM‐10; appendix 2D)
  • Lentiviral vector sample for titration, carrying GFP transgene (see protocol 1) and positive control (lentiviral supernatant already titered)
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2D)
  • 0.05% trypsin/EDTA (e.g., Invitrogen)
  • 1% (w/v) formaldehyde: dilute 1 ml of 37% formaldehyde (Sigma) in 36 ml PBS ( appendix 2D); store at 4°C
  • 6‐well tissue culture plates (e.g., BD Biosciences)
  • Fluorescence‐activated cell sorter (FACS, Becton Dickinson; with 488 nm excitation laser and green filter) or equivalent flow cytometer, and appropriate tubes
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified, 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 3: Biological Titration of Lentivectors by Quantitative PCR (QPCR)

  Materials
  • HeLa cells (ATCC cat. no. CCL‐2)
  • Lentiviral vector (LV) sample for titration (see protocol 1) and DNA from positive and negative control cells (see annotation to step 1)
  • DNAeasy Genomic DNA Extraction Kit (Qiagen)
  • Kit for preparing QPCR master mix (RT‐QP2X‐03; Eurogentec), including 2× reaction buffer
  • 10× TaqMan GAG set (see recipe)
  • 10× TaqMan HB2 set (see recipe)
  • MicroAmp 96‐well optical reaction plate (Applied Biosystems)
  • Optical caps (Applied Biosystems)
  • Centrifuge with microtiter plate carrier
  • Real‐time PCR machine (7700 Sequence Detector, Applied Biosystems)
  • Computer running ABI Prism software (Applied Biosystems) and Microsoft Excel
  • Additional reagents and equipment for transducing HeLa cells with lentivectors ( protocol 3)

Support Protocol 4: RCR Assay for LVS: Real‐Time Quantitative PCR (QPCR) Detection of Replication‐Competent Recombinants

  Materials
  • HeLa cells (ATCC cat. no. CCL‐2)
  • Lentiviral vector (LV) sample(s) and standards for titration (see protocol 1; also see last annotation to step 1 below)
  • 8E5 cells (ATCC cat. no. CRL‐8993)
  • DNAeasy Genomic DNA Extraction Kit (Qiagen)
  • Kit for preparing QPCR master mix (RT‐QP2X‐03; Eurogentec), including 2× reaction buffer
  • 10× TaqMan GAG set (see recipe) or 10× TaqMan POL set (see recipe)
  • 10× TaqMan HB2 set (see recipe)
  • MicroAmp 96‐well optical reaction plate (Applied Biosystems)
  • Optical caps (Applied Biosystems)
  • Centrifuge with microtiter plate carrier
  • Real‐time PCR machine (7700 Sequence Detector, Applied Biosystems)
  • Computer running ABI Prism software (Applied Biosystems) and Microsoft Excel
  • Additional reagents and equipment for transducing HeLa cells with lentivectors ( protocol 3)
CAUTION: ATCC recommends that 8E5 cells be handled in a P2 laboratory. Indeed, although they contain a full copy of noninfectious HIV, they can form syncytia with uninfected CD4+ cells.
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Figures

Videos

Literature Cited

Literature Cited
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   Recillas‐Targa, F., Pikaart, M.J., Burgess‐Beusse, B., Bell, A.C., Litt, M.D., West, A.G., Gaszner, M., and Felsenfeld, G. 2002. Position‐effect protection and enhancer blocking by the chicken beta‐globin insulator are separable activities. Proc. Natl. Acad. Sci. U.S.A. 99:6883‐6888.
   Salmon, P., Kindler, V., Ducrey, O., Chapuis, B., Zubler, R.H., and Trono, D. 2000a. High‐level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors. Blood 96:3392‐3398.
   Salmon, P., Oberholzer, J., Occhiodoro, T., Morel, P., Lou, J., and Trono, D. 2000b. Reversible immortalization of human primary cells by lentivector‐mediated transfer of specific genes. Mol. Ther. 2:404‐414.
   Wiznerowicz, M. and Trono, D. 2003. Conditional suppression of cellular genes: Lentivirus vector‐mediated drug‐inducible RNA interference. J. Virol. 77:8957‐8961.
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   Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L., and Trono, D. 1997. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15:871‐875.
   Zufferey, R., Dull, T., Mandel, R.J., Bukovsky, A., Quiroz, D., Naldini, L., and Trono, D. 1998. Self‐inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72:9873‐9880.
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