A fast replica plating technique for the isolation of post-integration mutants of the Moloney strain of murine leukaemia virus

Abstract

Seven temperature-sensitive (ts) mutants of Moloney murine leukaemia virus (Mo-MuLV) were isolated using a rapid, non-selective replica plating technique designed to select for post-integration mutants. Thymus-bone marrow (TB) cells, infected with mutagenized virus, were cloned and incubated at the non-permissive temperature (39 degrees C) for 10 days. The resulting colonies were screened for production of virus by replica plating supernatant from the "master" tray on to a second tray pre-seeded with fu-1 (a cell line derived from L8 myoblasts) indicator cells. The "master" tray was shifted to the permissive temperature (34 degrees C) for 48 h, then re-screened for virus production. Any colony on the "master" tray which produced syncytia-inducing virus at 34 degrees C but not at 39 degrees C was potentially producing a ts mutant. Preliminary characterization by shift-down experiments and scanning electron microscopy of three of the ts mutants isolated by this technique revealed a mutant blocked before budding, one blocked at an early stage in the budding process and one with a defect after release of the virus.