Abstract
Mouse genome contains thousands of copies of retrotransposon, intracisternal A-particle (IAP) DNA element, that is constructed from gag-pol gene sandwiched with two LTR sequence. All the cells have the potential of IAP-mediated retrotransposition that includes IAP cDNA synthesis from the RNA and its integration to the mouse genome. We have previously found that genomic rearrangement by insertions of new copies of the IAP cDNA frequently occur in acute myeloid leukemia cells of C3H/He mouse. Since there are large amounts of IAP-related DNA/RNA molecules in the cells, it is difficult to analyze the IAP cDNA that has the potential to convert genomic information. To study the behavior of IAP cDNA, we constructed a series of reporter genes to detect unique reverse transcript and stably introduced into the mouse cells.
Reversely transcribed cDNAs from the RNAs of the exogenously introduced plasmids were confirmed by the determination of the nucleotide sequence of the amplified products by PCR for the unique structure of the retrotransposition. We succeeded in the establishmeht of the methodology to determine relative amounts of the unique cDNA per cell using real-time PCR by simultaneous amplification of a reference plasmid that possessed both fragments of the unique sequence and a single-copy gene.
