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Dept of Molecular Pharmacology
Dept of Microbiology & Immunology

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Phoenix System > Episomes > Tetracycline Regulated

Phoenix helper-free retrovirus producer lines:

Phoenix is a second-generation retrovirus producer lines for the generation of helper free ecotropic and amphotropic retroviruses. The lines are based on the 293T cell line (a human embryonic kidney line transformed with adenovirus E1a and carrying a temperature sensitive T antigen co-selected with neomycin).

The unique feature of this cell line is that it is highly transfectable with either calcium phosphate mediated transfection or lipid-based transfection protocols-- up to 50% or higher of cells can be transiently transfected.

The lines were created by placing into 293T cells constructs capable of producing gag-pol, and envelope protein for ecotropic and amphotropic viruses. The lines offered advantages over previous stable systems in that virus can be produced in just a few days.

The lines have several other improvements over other approaches and this has led to their broad application worldwide:

  • We added the facility to monitor gag-pol production on a cell-by cell basis by introducing an IRES-CD8 surface marker downstream of the reading frame of the gag-pol construct. Thus, CD8 expression is a direct reflection of intracellular gag-pol and the stability of the producer cell population's ability to produce gag-pol can be readily monitored by flow cytometry.
  • For both the gag-pol and envelope constructs non-moloney promoters were used to minimize recombination potential.
  • Different promoters for gag-pol and envelope were used to minimize their inter-recombination potential.
  • Two cell lines were created, Phoenix-Eco and Phoenix-Ampho. Gag-pol was introduced with hygromycin as the co-selectable marker and the envelope proteins were introduced with diptheria resistance as the co-selectable marker.
  • Finally, a line is available that expresses only gag-pol; this line is termed Phoenix-gp. This line is available for further pseudotyping of retroviral virions with other envelope proteins such as gibbon ape leukemia virus envelope or Vesicular stomatitus VSV-G protein.
  • Both Phoenix-Eco and Phoenix-Ampho have been extensively tested for helper virus production and established as being helper-virus free.
  • Both lines are capable of carrying episomes for long-term stable production of retrovirus (LZRS).


Related links:

Tetracycline regulated retroviruses



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