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Identification of exons in a novel embryonal carcinoma locus using the GRAIL program
Oncology Reports
|May 20, 2011
Summary
Researchers used retroviral insertion to create a mutant embryonal carcinoma cell line. They identified expressed exons in novel genomic DNA using the GRAIL program, demonstrating its effectiveness for gene discovery.
Area of Science:
- Developmental Biology
- Genomics
- Cancer Research
Background:
- Embryonal carcinoma (EC) cells are crucial models for studying oncogenesis and mammalian development.
- A mutant EC cell line (NR1-6) was generated via retroviral insertion (Neo(r)) into NR1-0 cells.
- This mutant cell line exhibits unique morphological, tumorigenic, and differentiation characteristics.
Purpose of the Study:
- To characterize the genomic regions flanking a retroviral insertion in a unique embryonal carcinoma cell line.
- To identify potential genes or functional elements within novel genomic DNA.
- To evaluate the utility of the GRAIL program for exon prediction in unexplored genomic sequences.
Main Methods:
- Sequencing of approximately 18 kb of DNA flanking the retroviral insertion site.
- Bioinformatic analysis using GCG and BLAST to identify sequence homology.
- Application of the GRAIL (Gene Recognition and Analysis Internet Link) program for exon candidate identification.
- Validation of predicted exons using reverse transcriptase (RT)-PCR and subsequent sequencing.
Main Results:
- No homology to known genes was found in the flanking regions, although B1 repeat and CA/GT dinucleotide repeats were identified.
- The GRAIL program predicted 5 potential exon candidates with varying probabilities.
- RT-PCR and sequencing confirmed the expression of mRNA corresponding to two excellent and one marginal GRAIL-identified exon candidates.
- The sequenced mRNA was identical to the genomic DNA, indicating functional exons.
Conclusions:
- The GRAIL program is an efficient and reliable tool for identifying expressed exons within large, uncharacterized genomic DNA sequences.
- This study successfully identified novel expressed sequences in the vicinity of a retroviral insertion in EC cells.
- The findings contribute to understanding gene discovery in complex genomic regions and the relationship between oncogenesis and development.
