Contingent replication assay (CRA) procedure for rapid isolation of enhancers

Abstract

A rapid procedure for the isolation of functional enhancer sequences consists of the construction of a shotgun DNA library in SV40-based plasmid shuttle vectors which depend on an enhancer for replication, the replication in monkey (CVI) cells of those vectors into which an enhancer sequence was inserted, the selective cleavage of unreplicated vectors by DpnI and the recovery of the replicated vectors by transfection into Escherichia coli. We describe conditions for the fusion of protoplasts to CVI cells, under which conditions the probability of only one type of plasmid entering a cell is increased and thus complementation and rescue of enhancer-less plasmids are decreased. The effectiveness of the procedure is demonstrated by the recovery of enhancers from bovine papillomavirus and Moloney murine sarcoma virus.