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Versatile Reporter Vectors for Monitoring Viral Transduction

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

pFB-Luc Vector

Fig.3

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.

Conclusions

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
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