Retroviral vectors, by nature, infect and stably integrate into the genome of the host cell. Therefore, any cell line expressing the fluorescent protein marker from a retroviral vector is already an established stable cell line, unlike those from plasmid constructs. Coexpression of genes from different promoters can lead to competition between the promoters, and the eventual repression of one of the promoter elements. To eliminate this problem, our vectors use a single promoter to drive the production of IRES-containing bicistronic transcripts. The IRES allows cap-independent translation from an internal start site, resulting in stable coexpression of your protein of interest along with a fluorescent marker protein (Mountford and Smith, 1995). For expressing fluorescently tagged fusion proteins, use Retro-X Living Colors fusion vectors with DsRed-Monomer or AcGFP1.
Tech Note
Fluorescent retroviral expression vectors
- Efficient delivery of fusion proteins into hard-to-transfect cells
- Enhanced gene expression
- High-titer, self-inactivating retroviral backbone ensures robust expression
Introduction
Results
Coexpression of genes from different promoters
Our Retro-X IRES Living Colors vectors are designed to efficiently coexpress your gene of interest with our brightest red (DsRed-Express) or green (ZsGreen1) fluorescent markers. This is accomplished through the production of bicistronic transcripts that contain an internal ribosome entry site (IRES). The IRES allows independent translation of a second protein from a single transcript (Figure 1). These vectors contain all of the features necessary to efficiently deliver your gene of interest into even hard-to-transfect cells.
Figure 1. Schematic diagram of bicistronic mRNA translation. The IRES permits a protein of interest and a fluorescent protein to be independently translated from the same mRNA. SD = Splice Donor; SA = Splice Acceptor; Ψ = packaging signal; LTR = long terminal repeat.
Additional benefits to using these vectors include detection of the fluorescent marker by flow cytometry or fluorescence microscopy (Figure 2), allowing you to easily determine your transfection/transduction efficiency. Second, because both proteins are translated from a single transcript, the expression level of the easily detectable fluorescent marker allows you to approximate that of your gene of interest. Third, the fluorescent marker allows you to use flow cytometry to enrich for cells expressing your gene of interest. This is especially useful when introducing your gene of interest into hard-to-transfect cells (Figure 3.) Finally, you can rapidly titer the infectivity of your viral stocks by counting fluorescent cells or by using flow cytometry.
Figure 2. Retro-X IRES Living Colors vectors are ideal for visualizing infected cells. pRetroX-MetLuc-IRES-DsRed-Express (encoding Metridia secreted luciferase; 5) was transfected into GP2-293 cells, and the VSV-G-pseudotyped virus was harvested at 72 hr post-transfection. The viral stock was then used to infect HeLa cells (MOI = 10), and expression was visualized at 96 hr postinfection using fluorescence microscopy. MOI = multiplicity of infection.
Figure 3. Very high infection efficiencies can be achieved in hard-to-transfect cells. GP2-293 packaging cells were cotransfected with pVSV-G and either pRetroX-IRES-ZsGreen1 (negative control) or pRetroX-MetLuc-IRES-ZsGreen1 (encoding Metridia secreted luciferase; Yu et al. 2000). The resulting VSV-G pseudotyped viruses were used to infect Jurkat cells on plates coated with RetroNectin reagent. Expression of MetLuc and ZsGreen1 was measured by luminometry and flow cytometry, respectively, at 96 hr post-infection. Panel A. Analysis of Jurkat cells transduced with pRetroX-IRES-ZsGreen1. Panel B. Analysis of Jurkat cells transduced with pRetroX-MetLuc-IRES-ZsGreen1. MFI = Mean Fluorescence Intensity; RLU = Relative Light Units; M1 = Gate identifying cells that were not transduced with the vector; M2 = Gate identifying cells that were transduced with the vector.
Our vectors are based on the pMIN series of retroviral vectors, noted for their high titers, enhanced gene expression, and improved safety profiles (Yu et al. 2000). High titer production and enhanced gene expression result from the presence of highly optimized splicing machinery, and the MMLV LTR promoter (Figure 4). Improved safety profiles, on the other hand, result from the removal of all retroviral coding sequences, which reduces the likelihood that replication competent retrovirus will result from homologous recombination (Yu et al. 2000).
Figure 4. Retro-X IRES Living Colors vectors are ideal for high-level coexpression of your gene of interest with a fluorescent marker. GP2-293 packaging cells were cotransfected with pVSV-G and either pRetroX-IRES-ZsGreen1 (negative control), or pRetroX-MetLucIRES-ZsGreen1 (encoding Metridia secreted luciferase; Yu et al. 2000). The resulting VSV-G-pseudotyped viruses were harvested at 72 hr post-transfection. HeLa cells were then infected with either 1,000 µl, 100 µl, or 10 µl of the VSV-G-pseudotyped viral stocks (as indicated). Measurement of MetLuc (in relative light units, RLU) was performed at 48 and 72 hr post-infection, while the mean fluorescence intensity (MFI) from ZsGreen1 was measured at 72 hr post-infection. Viral titers were determined to be approximately 1x107 IFU/ml based on the percentage of fluorescent cells (data not shown). These results show that the pRetroXIRES-ZsGreen1 vector produces high titers and coexpresses high levels of protein.
Fusion protein studies
Retro-X Living Colors Fusion vectors are designed to facilitate delivery of distinct red and green fluorescent proteins that have been specifically engineered to provide outstanding performance when expressed as a fusion with your protein of interest (Gurskaya et al. 2003).
DsRed-Monomer and AcGFP1 proteins are ideal tools for monitoring gene expression and intracellular protein trafficking. Because of their distinct spectra, these fluorescent proteins can be used for multicolor labeling and direct visualization applications. Furthermore, due to their monomeric character, it is now possible to localize AcGFP1 and DsRed Monomer to compartments and structures that can only be targeted with non-oligomerizing fusion tags. Both DsRed-Monomer and AcGFP1 proteins are extremely stable and are ideal for subcellular localization studies. With these vectors, you can visualize biological processes as they occur and also easily track your protein of interest to a specific subcellular organelle or structure.
These vectors offer advantages beyond those of standard plasmids. Retroviral vectors, by nature, infect and stably integrate into the genome of the host cell. Therefore, any cell line expressing the fluorescent protein marker is already an established stable cell line. Constructing dual-color stable cell lines has never been easier (Figure 5).
Figure 5. Retro-X Living Colors fusion vectors are ideal for creating dual-color stable cell lines. pRetroQ-DsRed Monomer-Golgi and pRetroQ-AcGFP1-Tubulin constructs were transfected individually into GP2-293 cells, and the VSV-G pseudotyped virus was harvested at 48 hr post-transfection. These viral stocks were then used to co-infect HeLa cells, and expression was visualized at 48 hr post-infection using a fluorescent microscope.
Conclusions
Retro-X IRES Living Colors vectors are designed to efficiently coexpress your gene of interest with DsRed-Express or ZsGreen1 fluorescent markers. Retro-X Living Colors fusion vectors contain all of the features necessary to efficiently deliver your fluorescently tagged protein of interest into hard-to-transfect cell types. Both N- and C-terminal versions of the DsRed Monomer and AcGFP1 fluorescent proteins are available.
These vectors allow for easy detection of the fluorescent marker by flow cytometry or fluorescence microscopy to determine your transfection/transduction efficiency. Since both proteins are translated from a single transcript, the expression level of the easily detectable fluorescent marker allows you to approximate that of your gene of interest. In addition, the fluorescent marker allows you to use flow cytometry to enrich for cells expressing your gene of interest. This is especially useful when introducing your gene of interest into hard-to-transfect cells. Finally, you can rapidly titer the infectivity of your viral stocks by counting fluorescent cells or by using flow cytometry.
References
Gurskaya, N. G., et al. A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants. Biochem J. 373, 403–8 (2003).
Mountford, P. S. & Smith, A. G. Internal ribosome entry sites and dicistronic RNAs in mammalian transgenesis. Trends Genet. 11, 179–184 (1995).
Yu, S., Kim, J., Kim, S., High efficiency retroviral vectors that contain no viral coding sequences. Gene Ther. 7, 797–804 (2000).
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