Semliki Forest Virus and Sindbis Virus Vectors

Markus U. Ehrengruber1, Kenneth Lundstrom2

1 University of Zurich, Zurich, 2 Regulou Inc., Oberwil
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 12.2
DOI:  10.1002/0471142905.hg1202s33
Online Posting Date:  August, 2002
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Abstract

Semliki Forest virus (SFV) and Sindbis virus (SIN) are two, positive‐strand RNA viruses of the alphavirus genus. Vectors for both have been developed to express high levels of foreign genes in vitro and in vivo. describes the preparation of packaged SFV and SIN replicons by co‐electroporation of helper and vector RNA into baby hamster kidney (BHK)‐21 cells. describes the activation of packaged SFV replicons with a‐chymotrypsin. provides a method for the infection of hippocampal slices. is a technique for the infection of primary cultures of dispersed neurons with infectious SFV and SIN replicons. The describes a method for the cotransfection of in vitro‐transcribed vector and helper RNA into BHK‐21 cells. describes determining the titers of infectious SFV and SIN replicon stocks, and for metabolic labeling of infected cells.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Packaged SFV and Sin Replicons
  • Basic Protocol 2: Activation of Packaged SFV Replicons
  • Basic Protocol 3: Infection of Hippocampal Slices
  • Basic Protocol 4: Infection of Dispersed Neurons
  • Alternate Protocol 1: LIPID‐Mediated Contransfection of RNA
  • Support Protocol 1: Titer Determination
  • Support Protocol 2: Metabolic Labeling
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Packaged SFV and Sin Replicons

  Materials
  • Vector plasmid pSFV2gen (Invitrogen, upon request)
  • Helper plasmid pSFV‐Helper2 (Invitrogen)
  • Restriction endonucleases SpeI, NruI, and SapI (including corresponding buffers)
  • Vector plasmid pSINRep5 (Invitrogen) or pSINrep504 (Frolov and Schlesinger, , )
  • Linearized/nonlinearized helper plasmid DH‐BB or DH(26S)5′SIN (Invitrogen)
  • Restriction endonuclease XhoI (if the cDNA of interest contains an XhoI site, use either NotI or PacI to linearize pSINRep5) including corresponding buffer
  • 0.8% (w/v) agarose gel
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • 3 M sodium acetate ( appendix 2D)
  • 95% and 70% ethanol
  • recipe10× transcription buffer (SFV system, see recipe) or commercial 5× transcription buffer provided with the SP6 polymerase (SIN system)
  • 10 mM m7G(5′)ppp(5′)G (sodium salt; Pharmacia or New England Biolabs)
  • 50 mM dithiothreitol (DTT)
  • rNTP mix (10 mM rATP, 10 mM rCTP, 10 mM rUTP, 5 mM rGTP; Roche Molecular Biochemicals)
  • 10 to 50 U/µl RNase inhibitor (Roche Molecular Biochemicals)
  • 10 to 20 U/µl SP6 RNA polymerase (Roche Molecular Biochemicals, Invitrogen, Epicentre Technologies, or Promega)
  • Gel loading buffer ( appendix 2D)
  • Molecular weight marker (e.g., digested λ DNA)
  • BHK‐21 cells (ATCC #CRL‐6281, ∼80% confluent)
  • Phosphate‐buffered saline (PBS; appendix 2D), 37°C
  • Trypsin‐EDTA (0.5 mg/ml trypsin and 0.2 mg/ml EDTA in PBS)
  • recipeComplete BHK‐21 cell medium (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Two heating blocks or water baths (37°C and 80 to 90°C)
  • Sterile electroporation gap cuvettes (e.g., 0.2‐cm gap, Bio‐Rad)
  • Electroporator (e.g., Gene Pulser, Bio‐Rad)
  • Tissue culture flasks or dishes (24‐, 35‐, 60‐, or 100‐mm)
  • Plastic syringes (10‐ or 20‐ml) with attached 0.22‐µm sterile filters
  • Additional reagents and equipment for subcloning (CPMB UNIT ), preparation of plasmid DNA (unit 5.3), restriction endonuclease digestion (CPMB UNIT ), phenol/chloroform extraction and ethanol precipitation of DNA ( appendix 3C,spectrophotometric quantitation of RNA and DNA ( appendix 3D), agarose gel electrophoresis (unit 2.7), tissue culture ( appendix 3G), SDS‐PAGE for protein analysis ( appendix 3F), and autoradiography (CPMB APPENDIX )

Basic Protocol 2: Activation of Packaged SFV Replicons

  Materials
  • 20 mg/ml α‐chymotrypsin (Sigma)
  • Packaged SFV replicons (see protocol 1)
  • 10 mg/ml aprotinin (Sigma or Roche Molecular Biochemicals)
  • Phosphate‐buffered saline (PBS; appendix 2D), 37°C
  • BHK‐21 (ATCC #CRL‐6281), CHO (ATCC #CCL‐61), or HEK293 cells (ATCC #CRL‐1573) in 6‐ or 12‐well plates (70% to 80% confluent), plus the corresponding cell culture medium

Basic Protocol 3: Infection of Hippocampal Slices

  Materials
  • recipeCutting medium (see recipe), pre‐warmed to 37°C
  • Infectious SFV or SIN replicon stock
  • 95% ethanol
  • Hippocampal slice cultures (roller‐tube type)
  • recipeRoller tube culture medium (see recipe)
  • Glass capillaries (i.d. 1.5 mm, o.d. 1.17 mm; e.g., Clark capillaries, Harvard Apparatus)
  • Electrode puller
  • Autoclavable electrode holder
  • Micromanipulator (Narishige)
  • Metal plate containing a base for a 35‐mm petri dish
  • 3‐way valve
  • 1‐ml syringe
  • Plastic tubing (i.d. 1 mm, o.d. 3 mm)
  • 35‐mm plastic petri dishes
  • Dissection microscope
  • Microloader pipet tips (autoclaved; Eppendorf)
  • Forceps

Basic Protocol 4: Infection of Dispersed Neurons

  Materials
  • Infectious SFV or SIN replicons
  • BHK‐21 cell medium
  • 50% and 20% (w/v) sucrose solution
  • Ultracentrifuge tubes for Beckman SW 40 or SW 41 rotor (e.g., Beckman or Contron)
  • Ultracentrifuge with Beckman SW 40 or SW 41 rotor
  • Centriprep‐50 or Centriprep‐100 centrifugal concentrators (Amicon)
  • 50‐ml disposable plastic tubes (Falcon)
  • Low speed centrifuge (500 to 3000 × g), accommodating the 50‐ml plastic tubes
  • 1.5‐ml microcentrifuge tubes
  • 1‐ml disposable plastic pipet
  • Additional reagents and equipment for producing primary cultures of neurons in plastic petri dishes, multi‐well plates, or on coverslips, and preparation of the corresponding neuronal culture medium (e.g., Vicario‐Abejón, )

Alternate Protocol 1: LIPID‐Mediated Contransfection of RNA

  • Opti‐MEM I reduced‐serum medium (Invitrogen)
  • DMRIE‐C reagent (Invitrogen)

Support Protocol 1: Titer Determination

  • 100% methanol
  • 0.2% (w/v) gelatin in PBS (blocking solution)
  • Primary antibody directed against the recombinant protein
  • Secondary antibody coupled to detection system
  • 2.5% (w/v) DABCO (reduces fading of FITC) in Mowiol 4‐88 (Calbiochem)
  • Glass slides

Support Protocol 2: Metabolic Labeling

  • Infected or transfected cells (see protocol 1, protocol 22, protocol 44, and protocol 5 for cell lines and neurons)
  • recipeStarvation medium (see recipe)
  • >1000 Ci/mmol [35S]‐methionine (1 mCi/0.1 ml), added to starvation medium
  • recipeChase medium (see recipe)
  • recipeLysis buffer (see recipe)
  • recipe2× SDS‐PAGE sample buffer (see recipe)
  • Precast 10% (w/v) Tris‐glycine polyacrylamide gels (Novex/Invitrogen)
  • recipeFixing solution (see recipe)
  • Amplify solution (Amersham Pharmacia Biotech)
  • Hyperfilm‐MP (Amersham Pharmacia Biotech)
  • SDS‐PAGE apparatus
  • Power unit
  • Bio‐Rad gel dryer
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE; appendix 3F)
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Figures

Videos

Literature Cited

Literature Cited
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   Dryga, S.A., Dryga, O.A., and Schlesinger, S. 1997. Identification of mutations in a Sindbis virus variant able to establish persistent infection in BHK cells: The importance of a mutation in the nsP2 gene. Virology 228:74‐83.
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   Ehrengruber, M.U., Hennou, S., Büeler, H., Naim, H.Y., Düglon, N., and Lundstrom, K. 2001. Gene transfer into neurons from hippocampal slices: Comparison of recombinant Semliki Forest virus, adenovirus, adeno‐associated virus, lentivirus, and measles virus. Mol. Cell.Neurosci. 17:855‐871.
   Ehrengruber, M.U., Lundstrom, K., Schweitzer, C., Heuss, C., Schlesinger, S., and Gähwiler, B.H. 1999. Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proc.Natl. Acad. Sci. U.S.A. 96:7041‐7046.
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Key Reference
   Ehrengruber et al. 1999. See above.
  This paper applies both infectious SFV and SIN replicons in cultured rat hippocampal slices, compares the results, and shows that neurons are preferentially infected by both vectors.
Internet Resources
  http://www.ehs.psu.edu/biosafety/bmbl‐1.htm
  This site contains guidelines from the National Institutes of Health (NIH) and Center for Disease Control (CDC) on handling alphavirus and biosafety level 2 practices.
  http://www.microbiology.wustl.edu/sindbis/sin_genes
  This is an excellent site containing a lot of background information on the biology of Sindbis virus and its use as an expression vector.
  http://www.invitrogen.com/catalog.html
  This site contains information on the SFV and SIN systems (search the catalog for “SFV” or “Sindbis”). An older version of the SFV vector plasmid (pSEUM) is described, but the vector plasmid used herein (pSFV2gen) is available from Invitrogen upon request.
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