Introduction
Retroviral gene transfer is a technique for efficiently introducing stable, heritable genetic material into
the genome of any dividing cell type. With Moloney Murine Leukemia Virus (MMLV) - based
vectors, transduction efficiencies of >90% are achievable for most mitotic cell types, and the copy
number per cell can be easily controlled by varying the multiplicity of infection.
Current retroviral gene transfer technology is based on the coordinated design of packaging cell lines
and retroviral expression vectors. The packaging cell line usually contains the viral gag, pol, and env
genes, necessary for particle formation and replication, these genes are stably integrated into the
packaging cell genome. Retroviral expression vectors provide the packaging signal ψ+, transcription
and processing elements, and a target gene. Inserts of up to 6.5 kb can be efficiently packaged.
Transfection of the retroviral vector into a packaging cell line produces high-titer, replicationincompetent
virus (Table 1).
The viral env gene, expressed by the packaging cell line, encodes the envelope protein, which
determines the range of infectivity (tropism) of the packaged virus. Viral envelopes are classified
according to the receptors used to enter host cells (Table 1). Ecotropic virus can recognize a receptor
found on only mouse and rat cells. Amphotropic virus recognizes a receptor found on a broad range of
mammalian cell types. Pantropic virus, such as retrovirus pseudo-typed with the envelope
glycoprotein from the vesicular stomatitis virus (VSV-G), can infect both mammalian and nonmammalian
cells. Unlike other viral envelope proteins, VSV-G mediates viral entry through lipid
binding and plasma membrane fusion. However, stable expression of the VSV-G envelope protein is
toxic, pantropic virus is usually produced by transiently cotransfecting a retroviral expression vector
and pVSV-G into a pantropic packaging cell line expressing only the viral gag and pol genes.
Conventional NIH-3T3 based retroviral packaging cell lines and Phoenix packaging cell lines have
limited stability and produce low viral yields, mainly due to poor expression level of the retroviral
structure proteins (gag, pol, env) in the packaging cells. The Platinum Packaging Cell Lines, potent
retrovirus packaging cell lines based on the 293T cell line, were generated using novel packaging
constructs with an EF1α promoter to ensure longer stability and high-yield retroviral structure protein
expression (gag, pol, env). Platinum cells can be kept in good condition for at least 4 months in the
presence of drug selection, and can produce retroviruses with an average titer of 1 x 106 infectious
units/mL by transient transfection. In addition, replication competent retroviruses (RCR) are virtually
nonexistent because only coding sequences of viral structural genes are used, avoiding any
unnecessary retroviral sequences.
Cell Biolabs’ ES/EC Amphotropic Platinum Retrovirus Expression System provides a simple method
to generate RCR-free amphotropic retrovirus at high titer. The kit includes Plat-A packaging cells,
pMCs-Puro retrovirus expression vector and pMCs-GFP control vector. pMCs retroviral vector
includes hybrid LTRs containing elements from both MMLV and PCMV and is capable of expressing
genes in both EC and ES cells.