A LACZ-BASED VECTOR SYSTEM FOR THE RAPID DETECTION OF V(D)J RECOMBINASE ACTIVITY

Episomal vectors have been developed which are useful for studying V(D )J recombination both after transient transfections and in stably tran sfected cells. In contrast to recombination substrates previously desc ribed for transient assays, rearrangement of these vectors results in expression of beta-galactosidase which can be visualized directly in t he transfected cell, shortening the time required for the assay to 1-2 days instead of 3-4 days. When these substrates are stably integrated into a preB cell line, subclones are found which show no beta-galacto sidase staining, although the substrate is properly integrated, transc riptionally active and the transfectants still possess recombinase act ivity. This finding suggests that, at least in some chromosomal locati ons, transcription through a locus bearing recombination-signal sequen ces is not sufficient for V(D)J recombination. Using these same vector s, we estimate that the frequency with which V(D)J recombination-negat ive preB variants arise is less than 10(-4) per generation.