RESUMO
In order to identify key genes involved in the development of atherosclerotic lesions, differentially expressed genes in atherosclerotic plaques obtained from diet-induced hypercholesterolemic rabbit aorta were screened using the differential display (DD) RT-PCR technique. Aortic RNAs were isolated from rabbits fed cholesterol-supplemented (2% cholesterol in lab-chow, w/w) chow diet for 12 weeks, followed by the synthesis of cDNAs by reverse-transcription using 2-base anchored oligo (dT) (5'-T11VN) as 3'-primers. Synthesized cDNAs were amplified by PCR using arbitrary 10-mers as 5'-primers and the same 3'-primers used in the reverse-transcription. Amplified cDNAs sized between 0.2 to 0.5kb obtained from control and cholesterol-fed rabbit aortas were displayed on the 6% DNA-sequencing gel for comparisons. The cDNA bands showing distinctive differences in patterns of display or in density of the band were extracted from the gel. A total of 66 differentially displayed cDNAs was isolated and subjected to the reverse-Northern and Northern blot analyses in order to confirm the differences. Through the extensive confirming processes, three cDNAs were finally selected (designated CRGRA-1 through -3) and their nucleotide sequences were determined. Two of those (CRGRA-1 and -2) were determined to be up regulated and the other (CRGRA-3) was down-regulated by the cholesterol-feeding. Upon homology search on databases for the identification of the genes, the first cDNA (CRGRA-1) turned out to be a part of a novel gene, the second one (CRGRA-2) was homologous (82%) to the corresponding segment of mitochondrial NADH dehydrogenase subunits 4 gene, and the last one (CRGRA-2) was identified to be homologous (94%) to a segment of human small GTP-binding protein (Rab7) gene.
