Differentially Expressed Aortic Genes in Cholesterol-fed Rabbits.

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) RTPCR 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) (S'-TuVN) as 3'-primers. Synthesized cDNAs were amplified by PCR using arbitrary 10-mers as S'-primers and the same 3'primers used in the reverse-transcription. Amplified cDNAs sized between 0.2 to 0.5 kb 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-l through -3) and their nucleotide sequences were determined. Two of those (CRGRA-l and -2) were determined to be upregulated and the other (CRGRA-3) was downregulated by the cholesterol-feeding. Upon homology search on databases for the identification of the genes, the first cDNA (CRGRA-l) 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 subunit 4 gene, and the last one (CRGRA-3) was identified to be homologous (94%) to a segment of human small GTPbinding protein (Rab7) gene. [ABSTRACT FROM AUTHOR]