Determine retroviral transduction efficiency and gene expression in virtually any target cell line
Brenda Rogers Katherine Felts Peter Vaillancourt
We describe three new ViraPort retroviral reporter vectors: pFB-hrGFP Y, pFB-Neo-LacZ ,, and pFB-Luc , which can be used to determine transduction efficiency and gene expression in virtually any target cell line. To assay for expression, use simple fluorescence microscopy or FACS with pFB-hrGFP, carry out a b-galactosidase-activity assay or in situ staining with pFB-Neo-LacZ, or perform a luciferase activity assay with pFB-Luc. These control vectors are available as plasmid DNA as well as transduction-ready, replication-incompetent, VSV-G pseudotyped high-titer retroviral supernatants. The vectors are particularly useful as controls when using the ViraPort retroviral gene expression systems.
We recently introduced a set of human and rodent ViraPort retroviral cDNA expression libraries inserted into the MMLV-based high-titer retroviral vector pFB1,2. These complex cDNA libraries, part of our growing collection of ViraPort retroviral premade libraries, are easily and efficiently introduced into virtually any mitotic cell type for screening based on gene function. The insert capacity of pFB, our MMLV-based replication-defective pFB vector, is approximately 8.0 kb because there is no extraneous sequence between the LTRs. We excluded selectable markers or readily detectable reporter genes from the ViraPort retroviral gene expression systems to ensure that the largest cDNAs possible could be inserted.
Dispensing with marker g enes eliminates a direct method for measuring the infectious titer of the resulting retroviral supernatants for a given target cell line, and expression levels cannot be readily determined for the target of choice. To counteract this problem, we constructed and validated a set of high-titer retroviral reporter vectors based on the pFB vector, which can be used to assess transduction efficiency and readily assay for gene expression in infected cells lines. These retroviral reporters are available as plasmid DNA as well as transduction-ready, VSV-G pseudotyped high-titer retroviral supernatants.
We recently cloned and humanized the green fluorescent protein from the sea pansy Renilla reniformis,* (See page 85). Mammalian tissue culture cells transfected with plasmids harboring the humanized reniformis green fluorescent protein (hrGFP) downstream of the viral promoter were bright green (emission peak: 506 nm), which compares to that typically seen for the humanized, red-shifted A. victoria GFP (EGFP) (data not shown). The coding sequence for hrGFP was inserted between the EcoR I and Xho I sites in the pFB vector to produce the pFB-hrGFP vector (Figure 1A). The vector was then tested in retrovirally transduced cells. To produce virus, a 293-derived cell line was transiently transfected with pFB-hrGFP together with the gag-pol and VSV-G env expression vectors pVPack-GP and pVPack-VSV-G.3 (Figure 1B) Transfections were carried out using the Transfection MBS mammalian transfection kit (Stratagene), modified according to Pear et. al.4 Figure 2 shows that HeLa cells transduced with the virus were highly fluorescent and readily detectable by fluorescence microscopy. To dete rmine transduction efficiencies for the vector, each of five target cell lines were infected with serially diluted viral supernatant and, after a 2-day expression period, the transduction efficiency was determined by FACS. Titers were calculated2,3 for supernatant dilutions that fell within the linear dose:response range for transduction. Titers in excess of 107 cfu/ml were achieved for four out of the five cell lines tested (Table 1). For experiments in which cells were infected at a low multiplicity of infection (MOI) and, thus, harbor only single copies of the hrGFP provirus, fluorescence over background was readily detectable with this vector.
1.4 x 107
4.9 x 107
2.0 x 107
2.0 x 107
2.5 x 106
2.0 x 107
6.3 x 107
2.4 x 107
3.0 x 107
2.8 x 106
5.0 x 106
6.0 x 106
* Titer = (fraction of cells fluorescing over background) x (supernatant dilution/ml) x (2 x 105 cells infected). Titers for pFB-Neo-LacZ vector = (fraction blue cells / total # cells in field) x (supernatant dilution / ml) x (2 x 105 cells infected); titers represent the average for 3 randomly chosen fields for each dilution.
The use of b-galactosidase as a reporter in retroviral vectors has a number of advantages over other reporters. The enzymatic activity of b-galactosidase can be used as a quantitative measure of gene expression from the viral promoter in a cell line of choice. In addition, virally transduced cells can be visualized in situ by staining with X-gal, and viral titers may be determined both by counting blue cells using light microscopy or by FACS analysis of infected cells following incubation with a fluorescent b-galactosidase substrate. Additionally, by employing the pFB-Neo-LacZ vector, viral titers may also be determined by G418-resistant colony formation due to the presence of the neo m arker downstream of the b-galactosidase gene in a bicistronic, IRES-containing cassette (Figure 1A).
We first tested the vector by preparing virus (described above) and infecting CHO cells. Transduced cells were fixed and stained in situ with X-gal using the In Situ b-Galactosidase Staining Kit (Stratagene); the resulting transduced cells stained an intense blue (Figure 2). Viral titers were then determined by counting blue-stained cells as a percentage of the total number of cells visible in a microscopic grid. As indicated in Table 1, titers greater than 106 cfu/ml were achieved for CHO cells (the only cell line tested). In a separate experiment, equivalent titers were determined in parallel by both X-gal staining and selection of neomycin-resistant colony formation.2
For certain applications in which a rapid and sensitive assessment of gene expression efficiency from the viral promoter in a target cell of choice is required, luciferase is the reporter of choice. In Figure 3, NIH-3T3 cells infected with VSV-G pseudotyped virions made using pFB-Luc at very low MOIs show a dose-responsive luciferase readout. In addition, they display a significant signal above background at an MOI of 1:20,000, which represents an infection of 200,000 cells with approximately 10 virus particles.
Use ViraPort retroviral reporter vectors in conjunction with ViraPort retroviral premade libraries to reveal the efficiency of any retroviral transduction. When carrying out costly and time-consuming gene delivery experiments, where gene products are not readi ly detected and where the efficiency of infection and gene expression for cell lines is unknown, valuable information can be determined from using these vectors. These vectors are useful in the ViraPort retroviral premade libraries. Stratagene offers three different vectors for the greatest versatility. The standard control vector pFB-Luc is supplied with each ViraPort retroviral premade library, and the pFB Neo-LacZ and pFB-hrGFP vectors are available separately.