The Exontrap vector has an intrinsic splicing function, allowing selective cloning of exon sequences from large genomic eukaryotic DNA fragments. This new route for the identification of eukaryotic genes does not involve an initial isolation of cellular mRNA.
Thus, genes, which are not transcribed during certain life cycle stages, can also be identified. Exon/intron mapping is greatly facilitated, since for the determination of exon boundaries only the trapped exons have to be sequenced and compared to the known gene.
Also, an exon library can be derived and screened for cell type specific genes with labeled cDNA from a panel of differentiated cells.
The Exontrap function is based on a shuttle vector containing prokaryotic and eukaryotic genetic elements for replication in both, bacteria and cell cultures.
The vector contains a 5´ and 3´ exon separated by a 600 bp intron sequence, which contains a polylinker for cloning. The recombinant vector is transfected into eukaryotic cells (e.g. COS cells) and transcribed. Whether the insert contains an exon in the correct orientation is determined by restriction or sequence analysis.
The mRNA is processed, i.e. the intron sequences originating from the vector, as well as those being introduced, are removed. After total RNA isolation, the mature mRNA is reverse transcribed into cDNA using a specific primer complementary to a sequence of the bordering exon. The cDNA is amplified by PCR using specific primers, which create restriction sites for further subcloning.
- Search for new eukaryotic genes
- Identification of genes which are not transcribed into RNA during certain life cycle stages
- ntron/exon mapping
- Exon libraries for screening the genome with labeled cDNA
- Removing introns before sequencing
- Expression of eukaryotic genes