Retroviral vectors
Retroviruses are among the most intensely studied vectors utilized for virus-mediated gene transfer. These studies established the foundation of exploiting retroviral vectors as vehicles for efficient gene delivery into broad range of tissues and organs. The capacity of efficient integration into the host genome, ability to infect dividing cells and shuttle large genetic payloads, and maintenance of stable, long-term trans-gene expression are attributes that have brought retroviral vectors to the forefront of gene delivery and gene therapy. However, inability of transducing nondividing cells has restricted employment of retroviruses primarily for gene transfer into hematopoietic cells. As a hallmark, all members of retro/lentiviral vector family are capable of converting single-stranded RNA (ssRNA) of the retrovirus into dscDNA (dsDNA), which can be then stably integrated into the host genome and replicated along with it. As highly evolved parasites retroviruses act in concert with cellular host factors to ensure delivery of their genetic payload into the nucleus, where they exploit host machineries to fulfill replication and long-term expression.
The most common retroviral vectors utilized in our vector core facility are these based on gamma-retrovirus (prototype MLV, or MuLV (murine leukemia viruses)). These retroviral vectors are extremely efficient means to deliver an cDNA-of-interest to a wide range of mammalian cell types. They are by far the easiest and fastest means to deliver genes stably to mammalian cells. The additional advantage of retroviral vectors is that they can be easily adopted for the applications to deliver large libraries of genes, peptides, and RNAi tools.
Retroviral vectors can be generated using transient protocol of transfection (very similar to that used for lentiviral vector production), or via transfection into stable cell lines: Phoenix- MLV cells. (Both methods are commonly used at the facility).
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 cells have been created in Dr. Nolan’s laboratory (Stanford University) and based on the HEK293T cells. Phoenix cells 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.
Figure 1. Production of retroviral vector particles by transfection into Phoenix cells.
Pantropic Retroviral Expression System (Phoenix-GP): The Pantropic- Phoenix cell line is features the HK293T cells that stably-express gamma-retroviral gag and pol proteins. Upon transient transfection of pVSV-G the cell line expresses an envelope glycoprotein from the vesicular stomatitis virus that is not dependent on a cell surface receptor; instead, it mediates viral entry through lipid binding and plasma membrane fusion.
Phoenix cell lines (2nd generation) have several other improvements included 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 Phoenix-Eco and Phoenix-Ampho have been extensively tested for helper virus production and established as being helper-virus free.
The Duke Viral Vector Core also offers retroviral vector-packaging using Platinum Packaging Cell Lines to create amphotropic, ecotropic, or pantropic retrovirus (Cell Biolabs):
Plat-A (amphotropic) or Plat-E (ecotropic), which stably expresses both MMLV Gag-Pol and an amphotropic or ecotropic envelope protein, with a retroviral expression vector. These cells require transfection of only an expression vector to produce retrovirus. Pantropic retrovirus can be generated using Plat-GP Retroviral packaging cell line, which stably expresses MMLV Gag-Pol. This cell line can be supplemented with VSV-G pseudotype to generate retroviral vector that is capable of transducing broad-spectrum of cells.
Retroviral vectors can be also generated via transient-transfection protocols; such as calcium-phosphate or polyethylenimine (PEI)-based protocols (similar with production of lentiviral vectors). We use the naïve HEK293T cells to generate retroviral vectors transiently. The Vector Core has amphotropic, ecotropic and pantropic (VSV-G) envelops to generate the respective vectors. We have a collection of gag-pol packaging cassettes to package MLV, MSCV (Murine Stem Cell Virus), and other vector systems.
Legend:
Legend: transient protocol for retrovirus production.
To generate retroviral particles calcium-phosphate or polyethylenimine (PEI) transient transfection protocols are employed. Human embryonic kidney (HEK) 293T cells, expressing a polyomavirus-derived Large-T antigen used for transfection of three cassettes: packaging, envelope and expression cassettes. The Envelope cassette delivers envelope protein (often the vectors pseudotyped with Vesicular Stomatitis Virus G protein (VSV-G) in trans. Packaging cassette, encodes gag and pol proteins (see above). Gag-Pol genes supply all necessary structural and enzymatic proteins requiring for the viral life cycle. Third cassette that requires for generating retrovirus is an expression cassette that is flanked by retroviral LTRs and the packaging signal-sequence of retrovirus. The LTRs are necessary to integrate the therapeutic gene into the genome of the target cell, while the packaging signal- acts as a signal sequence and is necessary for packaging RNA with the reporter or therapeutic gene into viral particles.
Legend: transient protocol for retrovirus production.
Legend: To generate retroviral particles calcium-phosphate or polyethylenimine (PEI) transient transfection protocols are employed. Human embryonic kidney (HEK) 293T cells, expressing a polyomavirus-derived Large-T antigen used for transfection of three cassettes: packaging, envelope and expression cassettes. The Envelope cassette delivers envelope protein (often the vectors pseudotyped with Vesicular Stomatitis Virus G protein (VSV-G); Packaging cassette, encodes gag and pol proteins (see above). Gag-Pol genes supply all necessary structural and enzymatic proteins requiring for the viral life cycle. Third cassette that requires for generating retrovirus is an expression cassette that is flanked by retroviral LTRs and the packaging signal-sequence of retrovirus. The LTRs are necessary to integrate the therapeutic gene into the genome of the target cell, while the packaging signal- acts as a signal sequence and is necessary for packaging RNA with the reporter or therapeutic gene into viral particles.
