System > Episomes > Tetracycline
helper-free retrovirus producer lines:
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
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.
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.
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.
both the gag-pol and envelope constructs non-moloney promoters were
used to minimize recombination potential.
promoters for gag-pol and envelope were used to minimize their inter-recombination
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
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.
Phoenix-Eco and Phoenix-Ampho have been extensively tested for helper
virus production and established as being helper-virus free.
lines are capable of carrying episomes for long-term stable production
of retrovirus (LZRS).