A Simple and Reliable Method for Screening Retroviral Producer Clones Without Selectable Markers

Abstract

Simplified retroviral vectors that lack dominant selectable markers are being used with increasing frequency. These simplified vectors may offer a number of advantages over selectable marker-containing constructs, including potentially higher titers and less immunogenicity. However, the use of these vectors has been limited by the cumbersome experimental approaches in establishing and characterizing useful producer cell clones. To address this issue, a simple and reliable assay was developed to identify retroviral producer cell lines with or without dominant selectable markers. Producer cells were first generated by standard transfection/transduction and clones isolated by limiting dilution. Supernatant from each clone was then screened by RNA dot blot to identify the best producer clone candidates. The semiquantitative nature of the RNA dot blot assay was validated using a retroviral vector containing neomycin phosphotransferase (neo). Titers obtained by conventional G418-resistant colony forming units/ml (G418^R cfu/ml) assays strongly correlated with the values by RNA dot blot procedure. RNA dot blot results also correlated well with titers estimated by Southern analysis of HeLa cells transduced with supernatant from each clone. The RNA dot blot technique is a rapid (2 days) and reliable method to screen retroviral producer cells, thereby facilitating the generation and characterization of simplified retroviral producer cell clones. Retroviral vectors have been the most widely used gene transfer agents in gene therapy clinical trials. Most of these vectors contain dominant selectable markers to facilitate isolation and titration of candidate producer cell lines. However, inclusion of a second gene as a dominant selectable marker may have unwanted effects in gene therapy studies. First, the need to include multiple genes in a vector results in both larger and more complex retroviral genomes, which may have detrimental effects on titers. Second, in vivo expression of selectable markers of non-human origin (e.g., neomycin phosphotransferase) may induce immune responses that could lead to elimination of transgene-expressing cells. On the other hand, the characterization of virus-producing clones becomes much more laborious in the absence of selectable markers. Therefore, a simple and reliable assay has been developed to identify clones producing recombinant retrovirus that lack dominant selectable markers.