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 identification of high-titer retrovirus vector production by packaging cell clones. The procedure employs Trizol LS reagent to purify vRNA from packaging cell supernatants, a sensitive dot blot assay, and Phosphorlmager technology to quantify packaged viral genomes in 2 days. Experiments performed on viral supernatants of known biological titer demonstrated that the vRNA dot blot assay was extremely sensitive and that dot intensity correlated directly with viral titer. It is often necessary to analyze approximately 100 virus producing cell clones, making this method useful as a rapid screen to identify the highest virus producing clones. The vRNA dot blot assay consistently identified a subset of candidate 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 procedure greatly facilitates virus titration by: (1) rapidly eliminating the vast majority of low-titer producer cell clones; (2) accurately identifying the subset of candidate high-titer producer clones for further biological titration and assessment of the proviral genomic structure; and (3) reducing laborious tissue culture manipulations to a minimum. Furthermore, the reliance of this method on molecular detection makes it ideally suited for the isolation of high-titer clones lacking a drug selection marker.
Murdoch
B
B
Department of Genetics, Hospital for Sick Children, Toronto, Ontario, Canada.
Pereira
D S
DS
Wu
X
X
Dick
J E
JE
Ellis
J
J
D018014:Gene Transfer Techniques; D005821:Genetic Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D012367:RNA, Viral / Q000032:analysis*; D012190:Retroviridae / Q000235:genetics*
D012367:RNA, Viral