Cloning And Identification Of Hyperlipidemia And Atherosclerosis Associated Genes Of Rabbits By Combining Suppression Subtractive Hybridization And CDNA Microarray

Atherosclerosis (AS) is a multifactorial disease caused by the interaction of established or emerging risk factors with multiple predisposing genes that regulate AS-related processes. It has been estimated that more than 400 genes can regulate AS-related processes such as inflammation, endothelial function, coagulation and lipid, amino acid and carbohydrate metabolism. Although, overall, they are still insufficient to explain the heritable propensity to AS.The aim of the research was to identify differentially expressed genes in the hypercholesterolemia sensitive rabbit liver and hyperlipidemia insensitive rabbit liver. Male New Zeland white rabbits were fed with atherogenic diet for 20 weeks, and were divided into hypercholesterolemiasensitive (n=6, 31.92±5.61mmol/L) group and hyperlipidemia insensitive(n=3, 13.31±3.87mmol/L) group based on serum cholesterol level.Suppression subtractive hybridization (SSH), which combines subtractive hybridization with PCR, provided generation of a population of PCR fragments enriched for transcripts of high or low abundance from differentially expressed genes. We performed reciprocal cDNA collections of representative mRNAs specific to hypercholesterolemia sensitive rabbit liver versus hypercholesterolemia insensitive rabbit liver. To quantitate the expression levels, high density nylon cDNA array hybridization was optimized. We screened 800 suppressive subtraction hybridization cDNA clones by cDNA microarray and sequenced 121 of those showing the most marked expression differences. The gene expression findings were confirmed by real time quantitive RT-PCR. 47 new ESTs have been obtained (GenBankAccn: CA747377-CA747381, CB338050- CB338057, CB367790-CB367802, CB483519-CB483539). Malate dehydrogenase 2, NADH dehydrogenase subunit 2, Guanine nucleotide-binding proteins, heat shock protein 70, ubiquitin B, integrin and so on were found to be at least 1.5-fold up regulated in hypercholesterolemia sensitive rabbit. While the experssion of genes such as oxysterol 7alpha-hydroxylase, serum amyloid A, troponin and etc were decreased in hypercholesterolemia sensitive rabbits liver. We describe some genes that may be involved in...