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Biomedicum Genomics Oy
support@bmgen.fi
Haartmaninkatu 8
FI-00290 Helsinki
Finland
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Production of Recombinant Lentiviruses and Retroviruses
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Recombinant lentivirus particles are produced in Biomedicum Genomics by
co-transfecting a highly transfectable cell line with a transfer vector,
which carries the shRNA or cDNA of interest, together with packaging
constructs. The two packaging constructs produce the proteins important
for viral capsid structure, assembly and function as well as the proteins
that determines the viral tropism (see below). The virus particles will be
assembled in the cells and released to the culture supernatant. These
recombinant virus particles will only carry the sequences of transfer
vector in their genome and therefore, they can transduce the target cells
with high efficiency yet the target cells will not propagate the
infection. The target cells need to be completely virus-free before taking
out from the viruslab and this can be verified with the p24 test also
offered as service by Biomedicum Genomics.
Recombinant retroviruses are produced in a similar way, the main
difference in the protocol being specific packaging cells. Retroviral
packaging cells contain stably integrated the backbone vector and the
envelope vector. Therefore, only the transfer vector needs to be
transfected to the packaging cells in order to produce recombinant
retrovirus.
Viral tropism and pseudotyping
The envelope proteins of retro- and lentiviruses determine the host cell
range (viral tropism). There are several options for envelope proteins,
since they are encoded by separate vector in the lentiviral systems and in
the retroviral systems the envelope is selected by choosing a desired
packaging cell line. The selection of an envelope protein to determine the
viral tropism is called pseudotyping. Biomedicum Genomics pseudotypes
recombinant lentiviruses with VSV-G, which allows infection of broad range
of mammalian and non-mammalian host cells including human, primate, mouse,
rat, hamster, and fish.
In addition to lentiviral particles, Biomedicum Genomics also produces
Moloney mouse leukemia virus (MMLV) and mouse stem cell virus (MSCV)
vector based retroviruses pseudotyped with ecotropic or amphotropic
envelope. Ecotropic retroviruses infect mouse and rat cells but not human
cells. Amphotropic viruses have broader host range infecting for example
human, primate, mouse, rat, and rabbit cells (for full list of host cells,
contact Biomedicum Genomics).
Recombinant lentiviral and retroviral particles are generally produced in
the biosafety level 2 containment. Regarding production and handling of
the recombinant lentiviral and retroviral particles, please adhere to the
rules and recommendations of your host institute.
Principles of recombinant lentivirus production
Biomedicum Genomics uses a standard three-plasmid lentiviral system to
produce high-titer VSV-G pseudotyped lentiviral particles. The recombinant
lentivirusparticles are produced by transient transfection of the
gene/shRNA transfer vector along with two packaging plasmids to an
optimized derivative of 293T cells. At the time of transfection, the
confluency of the cells should be approximately 50-60%. The 293T and
derivative cells are highly transfectable and can be efficiently
transfected by many standard methods, including liposomes and Calcium
Phosphate. BMGen recommends e.g. Lipofectamine (Invitrogen) or the
cationic polymer jetPEI™ (polyplus) transfection reagent. After
transfection, the cells are incubated for 4h at 37°C, after which the
medium is refreshed.
After 72h incubation, the virus-containing supernatant is collected and
filtered through 0,45 um PES filter. Virus supernatant is then aliquoted
in cryovials and stored in -80°C, or concentrated by
ultrasentrifugation.
Titer of the lentiviral supernatant is measured by ELISA p24 test, which
is carried out for both concentrated and unconcentrated samples.
With every virus batch produced, Biomedicum Genomics uses internal
controls to ensure the high quality of the process.
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