CLONE-SPECIFIC HIGH-FREQUENCY RETROTRANSPOSITION OF A RECOMBINANT VIRUS CONTAINING A VL30 PROMOTER IN SV40-TRANSFORMED NIH3T3 CELLS

A recombinant virus, containing the promoter of a VL30 LTR and tagged with the neomycin gene as a selection and indicator marker, was constr ucted to investigate transposition events in NIH3T3 cells after SV40 t ransformation. This retroviral construct was transfected into psi/CRE packaging cells, and pseudovirions were used to infect NIH3T3 cells. C lones resistant to G418 bearing single-copy integrations of the recomb inant virus were isolated and transformed by SV40 virus. Transposition s were detected through RFLPs with a neomycin probe and 'retrotranspos ition' was further confirmed by inverse PCR and DNA sequencing of tran sposed and parental copies. We found that: (1) retrotransposition of t his recombinant virus occurred with a high frequency in a parental clo ne transformed with SV40 virus suggesting that the frequency of retrot ransposition depended on the initial site of provirus integration; (2) the transposition frequency was independent of the transcription leve l of the recombinant construct; and (3) analysis of transposition-posi tive transformants showed that the high transposition frequency appear ed to be associated with the induction of endogenous reverse transcrip tases. (C) 1998 Elsevier Science B.V. All rights reserved.