A SIMPLE AND RELIABLE METHOD FOR SCREENING RETROVIRAL PRODUCER CLONESWITHOUT SELECTABLE MARKERS

Simplified retroviral vectors that lack dominant selectable markers ar e being used with increasing frequency. These simplified vectors may o ffer a number of advantages over selectable marker-containing construc ts, including potentially higher titers and less immunogenicity. Howev er, the use of these vectors has been limited by the cumbersome experi mental approaches in establishing and characterizing useful producer c ell clones. To address this issue, a simple and reliable assay was dev eloped to identify retroviral producer cell lines with or without domi nant selectable markers. Producer cells were first generated by standa rd transfection/transduction and clones isolated by limiting dilution. Supernatant from each clone was then screened by RNA dot blot to iden tify the best producer clone candidates. The semiquantitative nature o f the RNA dot blot assay was validated using a retroviral vector conta ining neomycin phosphotransferase (neo). Titers obtained by convention al G-418-resistant colony forming units/ml (G418(R) cfu/ml) assays str ongly correlated with the values by RNA dot blot procedure. RNA dot bl ot results also correlated well with titers estimated by Southern anal ysis of HeLa cells transduced with supernatant from each clone. The RN A dot blot technique is a rapid (2 days) and reliable method to screen retroviral producer cells, thereby facilitating the generation and ch aracterization of simplified retroviral producer cell clones.