An Exon Trapping System Providing Size Selection of Spliced Clones and Facilitating Direct Cloning

Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have developed an exon trapping procedure based on the use of vector pETVSD2. Cosmid DNA is partially digested and cloned in pETV-SD2. DNA of an entire library of subclones is introduced into COS-1 cells and transiently expressed. RNA is isolated and vector-derived transcripts are amplified by RT-PCR. Cloning of the RT-PCR products, which contain a ColEI-origin of replication and a supF marker, is established by NotI digestion and intramolecular circularisation. Due to their shorter length, spliced clones are preferentially amplified and cloned.