A RAPID SCREENING-PROCEDURE FOR THE IDENTIFICATION OF HIGH-TITER RETROVIRUS PACKAGING CLONES

We have developed a viral RNA (vRNA) dot blot assay for rapid identifi cation of high-titer retrovirus vector production by packaging cell cl ones. The procedure employs Trizol LS reagent to purify vRNA from pack aging cell supernatants, a sensitive dot blot assay, and Phosphorlmage r technology to quantify packaged viral genomes in 2 days. Experiement s performed on viral supernatants of known biological titer demonstrat ed that the vRNA dot blot assay was extremely sensitive and that dot i ntensity correlated directly with viral titer. It is often necessary t o analyze approximately 100 virus producing cell clones, making this m ethod useful as a rapid screen to identify the highest virus producing clones. The vRNA dot blot assay consistently identified a subset of c andidate high-titer producer cell clones. In three independent screens the supernatant with the highest biological titer was produced by one of the previously defined candidate high-titer producer clones. Our p rocedure greatly facilitates virus titration by: (1) rapidly eliminati ng the vast majority of low-titer producer cell clones; (2) accurately identifying the subset of candidate high-titer producer clones for fu rther biological titration and assessment of the proviral genomic stru cture; and (3) reducing laborious tissue culture manipulations to a mi nimum. Furthermore, the reliance of this method on molecular detection makes it ideally suited for the isolation of high-titer clones lackin g a drug selection marker.