Preparation of a Specific Retrovirus Producer Cell Line

Constance Cepko1

1 Havard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 9.10
DOI:  10.1002/0471142727.mb0910s36
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Establishing a cell line that produces high levels of a specific retrovirus construct involves several steps. First, the retroviral construct must be stably introduced into an appropriate packaging cell line. This can be accomplished either directly by transfection or by transfection to produce a transient virus stock followed by cross‐infection of this stock into a separate packaging line; procedures for both approaches are described in this unit. After stable lines are produced, they must be characterized to identify lines that produce high titers of virus with an appropriate structure. A drug selection protocol (the most common method for determining virus titer) is provided along with a sample calculation of BAG virus titer. Sometimes, a short and direct method of estimating virus titer is available when the virus encodes a histochemically detectable gene such as lacZ. For this case, an Xgal staining protocol is described. Similarly, a protocol is provided to estimate the levels of virus being produced by a given cell line by quantitating any specific product of the virus (e.g., RNA) present within the producer line.

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

Table of Contents

  • Basic Protocol 1: Introduction of a Retrovirus Vector into a Packaging Cell Line
  • Basic Protocol 2: Determination of Viral Titer: Identification of Producer Clones Making High‐Titer Virus
  • Support Protocol 1: Sample Calculation of Titer for BAG Virus
  • Alternate Protocol 1: Rapid Evaluation of Producer Colonies
  • Support Protocol 2: XGAL Staining of Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Introduction of a Retrovirus Vector into a Packaging Cell Line

  Materials
  • Appropriate packaging cell line(s) (unit 9.9) with appropriate medium
  • Retrovirus plasmid DNA
  • recipeHEPES‐buffered saline (HeBS; see recipe)
  • 2 M CaCl 2
  • Medium, without and with serum
  • recipeHeBS containing 15% (v/v) glycerol (HeBS/glycerol)
  • 800 µg/ml (100×) polybrene in distilled H 2O, filter sterilized (store at −20°C)
  • 10% to 15% (v/v) dimethylsulfoxide (DMSO)
  • 10‐cm or 6‐cm dishes
  • 24‐well or 6‐well tissue culture dishes
  • Cloning cylinders (unit 16.23).
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All incubations involving tissue culture cells should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.

Basic Protocol 2: Determination of Viral Titer: Identification of Producer Clones Making High‐Titer Virus

  • Target cell line (e.g., NIH 3T3 fibroblasts)
  • Virus stock (see protocol 1; e.g., Ψ2 BAG supernatant)
  • Xgal (see protocol 5), or G418 or other selection drug (e.g., unit 9.5)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All incubations involving tissue culture cells should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.

Support Protocol 1: Sample Calculation of Titer for BAG Virus

  Materials
  • Cells infected or transfected with a lacZ‐encoding virus (see protocol 1)
  • PBS ( appendix 22)
  • Fixative solution: recipe0.05% glutaraldehydeor recipe2% paraformaldehyde solution (see reciperecipes)
  • recipeXgal solution (see recipe)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All incubations involving tissue culture cells should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bender, M.A., Palmer, T.D., Gelinas, R.E., and Miller, A.D. 1987. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J. Virol. 61:1639‐1646.
   Cepko, C.L., Roberts, B.E., and Mulligan, R.E. 1984. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell 37:1053‐1062.
   Cosset, F.‐L., Legras, C., Chebloune, Y., Savatier, P., Thoraval, P., Thomas, J.L., Samarut, J., Nigon, V.M., and Verdier, G. 1990. A new avian leukosis virus (ALV)–based packaging cell line using two separate transcomplementing helper genomes. J. Virol. 64:1070‐1078.
   Graham, F.L. and van der Eb, A.J. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:446‐467.
   Hughes, S.H., Greenhouse, J.J., Petropoulos, C.J., and Sutrave, P. 1987. Adaptor plasmids simplify the insertion of foreign DNA into helper‐independent retroviral vectors. J. Virol. 61:3004‐3012.
   Korman, A.J., Frantz, J.D., Strominger, J.L., and Mulligan, R.C. 1987. Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc. Natl. Acad. Sci. U.S.A. 84:2150‐2154.
   Miller, A.D. and Buttimore, C. 1986. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell. Biol. 6:2895‐2902.
   Morgan, B.A. and Fekete, D.M. 1996. Manipulating gene expression with replication competent retroviruses. Methods Cell Biol. 51:185‐218.
   Parker, B.A. and Stark, G.R. 1979. Regulation of simian virus 40 transcription: Sensitive analysis of the RNA species present early in infections by virus or viral DNA. J. Virol. 31:360‐369.
   Price, J., Turner, D., and Cepko, C. 1987. Lineage analysis in the vertebrate nervous system by retrovirus‐mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 84:156‐160.
   Rein, A., Schultz, A.M., Bader, J.P., and Bassin, R.H. 1982. Inhibitors of glycosylation reverse retroviral interference. Virology 119:185‐192.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library