Gene Delivery Using Helper Virus–Free HSV‐1 Amplicon Vectors

Cornel Fraefel1

1 Institute of Virology, Zurich
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.12
DOI:  10.1002/0471142905.hg1212s33
Online Posting Date:  August, 2002
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Herpes simplex virus type 1 (HSV‐1)‐based amplicon vectors contain only ˜1% of the viral genome. Consequently, replication and packaging of amplicons depend on helper functions provided either by replication‐defective mutants of HSV‐1 (helper viruses) or by replication‐competent, but packaging‐defective, HSV‐1 genomes. Sets of cosmids that overlap and represent the entire HSV‐1 genome can form, via homologous recombination, circular replication‐competent viral genomes, which give rise to infectious virus progeny. If the DNA cleavage/packaging signals are deleted, reconstituted virus genomes are not packageable, but still provide all the helper functions required for the packaging of cotransfected amplicon DNA. Resulting stocks of packaged amplicon vectors are free of contaminating helper virus. The basic protocol describes the cotransfection of amplicon and cosmid DNA into 2‐2 cells and the harvesting of packaged vector particles. Support protocols describe preparing cosmid DNA and methods for determining the titers of amplicon stocks.

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Table of Contents

  • Basic Protocol 1: Preparation of Helper Virus–Free Amplicon Stocks
  • Support Protocol 1: Preparation of HSV‐1 Cosmid DNA for Transfection
  • Support Protocol 2: Titration of Amplicon Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Helper Virus–Free Amplicon Stocks

  • 2‐2 cells (Smith et al., 1992)
  • Dulbecco's modified Eagle medium (Life Technologies) with 10% and 6% fetal bovine serum (DMEM/10% FBS and DMEM/6% FBS)
  • G418 (Geneticin; Life Technologies)
  • 0.25% trypsin/0.02% EDTA (Life Technologies)
  • Opti‐MEM I reduced‐serum medium (Life Technologies)
  • PacI‐digested cosmid DNA of set C6Δa48Δa (see protocol 2)
  • HSV‐1 amplicon DNA (maxiprep DNA isolated from E. coli)
  • LipofectAMINE reagent (Life Technologies)
  • 10%, 30% and 60% (w/v) sucrose in PBS
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • 75‐cm2 tissue culture flasks
  • Humidified 37°C, 5% CO 2 incubator
  • 60‐mm‐diameter tissue culture dishes
  • 15‐ml conical centrifuge tubes
  • Dry ice/ethanol bath
  • Probe sonicator
  • 0.45‐µm syringe‐tip filters (use Sarstedt polyethersulfone membrane filters)
  • 20‐ml disposable syringes
  • 30‐ml centrifuge tubes (Beckman Ultra‐Clear 25 × 89 mm and 14 × 95 mm)
  • Sorvall SS‐34 rotor
  • Fiber‐optic illuminator
  • Ultracentrifuge with Beckman SW28 and SW40 rotors

Support Protocol 1: Preparation of HSV‐1 Cosmid DNA for Transfection

  • E. coli clones of HSV‐1 cosmid set C6Δa48Δa,which includes cos6Δa, cos14, cos28, cos48Δa, and cos56 (see Fig. )
  • recipeSOB medium containing 50 µg/ml ampicillin (SOB/amp; see recipe)
  • Dimethyl sulfoxide (DMSO)
  • Plasmid Maxi Kit (Qiagen), which includes Qiagen‐tip 500 columns and buffers P1, P2, P3, QBT, QC, and QF (prewarm buffer QF to 65°C)
  • Isopropanol
  • 70% (v/v) ethanol
  • TE buffer, pH 7.5 ( appendix 2D)
  • Restriction endonucleases DraI, KpnI, and PacI
  • High‐molecular‐weight DNA standard (Life Technologies)
  • 1‐kb DNA ladder (Life Technologies)
  • Electrophoresis‐grade agarose
  • recipeTAE electrophoresis buffer (see recipe)
  • 1 mg/ml ethidium bromide in H 2O
  • 25:24:1 (v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C3C)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 100% ethanol
  • 3 M sodium acetate, pH 5.5 ( appendix 2D)
  • 17 × 100−mm graduated snap‐cap tubes (e.g., Falcon 2059), sterile
  • Sorvall GSA and SS‐34 rotors
  • 65° and 37°C water baths
  • 250‐ml polypropylene centrifuge tubes
  • 30‐ml centrifuge tubes

Support Protocol 2: Titration of Amplicon Stocks

  • Vero (clone 76; ECACC #85020205), BHK (clone 21; ECACC #85011433), or 293 (ATCC #1573) cells
  • DMEM (e.g., Life Technologies) supplemented with 10% and 2% FBS (DMEM/10% FBS and DMEM/2% FBS)
  • PBS ( appendix 2D)
  • Samples collected from vector stocks (see protocol 1, steps , , or )
  • recipe4% (w/v) paraformaldehyde solution, pH 7.0 (see recipe)
  • recipeX‐gal staining solution (see recipe), recipeGST solution (see recipe), or appropriate primary and secondary antibodies
  • 24‐well tissue culture plates
  • Humidified 37°C, 5% CO 2 incubator
  • Inverted fluorescence microscope
  • Inverted light microscope
NOTE: The titers expressed as transducing units per milliliter (t.u./ml) are relative and do not necessarily reflect numbers of infectious vector particles per milliliter. Factors influencing relative transduction efficiencies include: (1) the cells used for titration, (2) the promoter regulating the expression of the transgene, (3) the transgene, and (4) the sensitivity of the detection method.NOTE: All cell culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise stated.
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Literature Cited

Literature Cited
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   Cunningham, C. and Davison, A.J. 1993. A cosmid‐based system for constructing mutants of herpes simplex virus type 1. Virology 197:116‐124.
   Fraefel, C., Song, S., Lim, F., Lang, P., Yu, L., Wang, Y., Wild, P., and Geller, A.I. 1996. Helper virus‐free transfer of herpes simplex virus type 1 plasmid vectors into neural cells. J. Virol. 70:7190‐7197.
   Fraefel, C., Jacoby, D.R., Lage, C., Hilderbrand, H., Chou, J.Y., Alt, F.W., Breakefield, X.O. and, Majzoub, J.A. 1997. Gene transfer into hepatocytes mediated by helper virus‐free HSV/AAV hybrid vectors. Mol. Med. 3:813‐825.
   Fraefel, C., Breakefield, X.O., and Jacoby, D.R. 1998. HSV‐1 amplicon. In Gene Therapy for Neurological Disorders and Brain Tumors (E.A. Chiocca and X.O. Breakefield, eds.) pp. 63‐82. Humana Press, Totowa, N.J.
   Geller, A.I. and Breakefield, X.O. 1988. Defective HSV‐1 vector expresses Escherichia coli β‐galactosidase in cultured peripheral neurons. Science 241:1667‐1669.
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