Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.

Abstract

We have previously shown that the G protein ofvesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracyclinedependent and can be modulated by 18-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.Retroviral vectors derived from Moloney murine leukemia virus have been used extensively to transduce genes into mammalian cells (1, 2). However, problems such as the inability to infect quiescent cells, low virus titers, and relatively poor infectivity in human cells severely limit the application of retroviral vectors in gene transfer. To overcome some of these problems, we have generated retroviral vectors pseudotyped with the G protein of vesicular stomatitis virus (VSV-G) (3-5). We have shown that the pseudotyped retroviral vectors have an expanded host range and are able to infect many cell types with a greater efficiency than traditional amphotropic retroviral vectors (3-6). Moreover, the pseudotyped virus can be concentrated by ultracentrifugation to titers greater than 109 colony forming units/ml (4). These unique properties of the VSV-G pseudotyped virus not only extend the use of retroviral vectors for genetic studies in previously inaccessible species, but also facilitate more efficient preclinical and clinical studies of the potential for human gene therapy.However, the generation of stable packaging cell lines for pseudotyped retroviral vectors has been difficult because of the toxicity of VSV-G in most mammalian cells (4, 5). To overcome this problem, we have modified the tetracyclineinducible system (7) to regulate the expression of VSV-G. In that system, a chimeric transcription factor, tTA, was produced by combining the repressor of Escherichia coli tetracyclineresistance operon (tetR) and the activation domain of virion protein 16 (VP16) of herpes simplex virus (HSV). This chimeric transactivator can regulate gene expression from a minimal promoter linked to multiple copies of tetO, the binding site for the tetracycline repressor. In the absence of tetracycline, tTA binds the tetO site and activates transcription from the tetO-containing promoter. In the presence of tetracycline, interaction of tTA with the tetO is prevented by its binding to tetracycline, thereby prevents transcription.While the expression of several genes has been reported to be inducible using this system (7-11), establishment of cell lines stably expressing tTA has been difficult, presumably because the activation domain of VP16 is capable of squelching general cellular transcription when expressed in l...

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“…of 5:1 colony-forming units per cell in the presence of Polybrene (8 g/ml). For mock-infected control cells, CHO cells were infected with LacZ VSV-G pseudotyped retrovirus (LZRNL) that generated from the same pLRNL retroviral backbone (provided by Dr. Miyanohara) (14). As a preliminary experiment to determine the effect of different m.o.i.s on infection efficiency, the cells were infected with LacZ retrovirus at an m.o.i.…”

“…The method for generation of pseudotyped retrovirus with G-glycoprotein of the vesicular stomatitis virus (VSV-G) envelope has been previously described (14). Briefly, with calcium phosphate coprecipitation LFDRNL cDNA and an expression plasmid for amphotropic envelope gene were transfected into a packaging cell line 293GP cells that express Moloney murine leukemia virus gag-pol gene.…”

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“…Additional advantages of VSV-G-pseudotyped lentivirus are the higher viral titers compared to those obtained with other envelope proteins and the improved vector particle purification due to increased stability of the virus. However, when used in high concentrations, lentiviral vectors bearing VSV-G may exert cytotoxic effects (Chen et al, 1996). Fortunately, this drawback can be overcome either by improving purification of the lentiviral particles using gradient centrifugation to eliminate unincorporated transgene particles (Ricks et al, 2008) or by using other proteins for pseudotyping.…”