A rapid test for V(D)J recombinase activity.

We describe a rapid and easy method to assay lymphoid V(D)J recombinase activity in cultured cell lines using a self-replicating recombination substrate. The substrate for recombination is a pBluescript based (Stratagene) plasmid (pBlueRec, see Figure la) in which the lacZ gene is interrupted by a DNA fragment flanked by consensus recombination signal sequences (Fig. lb). Site specific recombination will lead to deletion of the interrupting sequences restoring the correct reading frame of LacZ in one out of three cases due to imprecise excision. Cells (3 x 10) were transfected with 150 ng of pBlueRec using DEAE-dextran as described (1) or by electroporation (2) with 5 /ig of pBlueRec. 48 hours after transfection the cells were collected and the plasmid recovered using the rapid alkaline lysis method (3). In order to analyse only those molecules which had entered the nucleus we performed a Dpnl digestion, which eliminates plasmids which have not replicated and have thus conserved the bacterial dam methylation, prior to transformation. Recovered plasmid was introduced into XL 1-blue (Stratagene) bacteria and transformed cells were plated on LB agar plates containing Xgal (80 /tg/ml), IPTG (150 /tM), ampicillin (100 /tg/ml) and tetracycline (10 jig/ml). In frame rearrangement of the recombination substrate gives rise to blue colonies. Transfection experiments with recombination competent preB cells (22D6, NFS70, 7853) gave both blue and white clones, whereas transfection of a mature B-cell line (X63 Ag8) gave only white colonies. The rearrangement frequency (blue clones x3/total number of clones x 100) varied between 1.6 and 6.8% depending on the pre-B cell line. Sequencing showed that blue clones correspond to site specific V(D)J recombination with characteristic deletions and/or N region insertions (Fig. lc). The assay we describe uses extrachromosomal DNA and is therefore easier and less time consuming than the use of stably integrated recombination substrates (4). This method has the added advantage over previously reported extrachromosomal substrates (1) in that the results are read on a single plate making it more practical and reducing the experimental error.