( enes eliminates a direct method for mea...)Versatile Reporter Vectors for Monitoring Viral Transduction

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.

pFB-hrGFP Vector

Fig.1

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
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