INCREASED EXON-TRAPPING EFFICIENCY THROUGH MODIFICATIONS TO THE PSPL3SPLICING VECTOR

Exon trapping allows for the rapid identification and cloning of codin g regions from cloned eukaryotic DNA. In preliminary experiments, we o bserved two phenomena which limited the exon-trapping efficiency of pS PL3-based systems. The first factor that affected performance was reve aled when we found that up to 50% of the putative trapped exons contai ned sequences derived from the intron of the pSPL3 trapping vector. Re moval of the DNA sequences responsible for the cryptic splice event fr om the original splicing vector resulted in a new vector, pSPL3B. We d emonstrate that pSPL3B virtually eliminates pSPL3-only spliced product s while maximizing the proportion of exon traps containing genomic DNA (>98%). The other step which impacted performance was our observation that a majority of the ampicillin-resistant (Ap(R)) clones produced a fter shotgun subcloning from Ap(R) cosmids into pSPL3 were untrappable , pSPL3-deficient, recircularized cosmid vector fragments. Replacement of the pSPL3 Ap(R) gene with the Cm-R cassette encoding chloramphenic ol (Cm) acetyltransferase enabled selection for only pSPL3-containing Cm-R clones. We show a 30-40-fold increase in the initial subcloning e fficiency of cosmid-derived fragments with pSPL3-CAM, when compared to pSPL3. The collective vector alterations described improve the overal l exon-trapping efficiency of the pSPL3-based trapping system.