cDNA RDA在类似普通2型糖尿病大鼠肾脏组织基因差异表达筛查中的应用

应用代表性差异分析 (cDNARDA)技术 ,对类似普通 2型糖尿病大鼠肾脏组织基因差异表达进行筛查 ,初步探讨类似普通 2型糖尿病大鼠肾脏损害发病的分子机制 .首先以类似普通 2型糖尿病大鼠肾脏组织作为实验组 (Tester) ,正常大鼠肾脏作为对照组或驱动组 (Driver)通过cDNARDA进行基因差异表达筛查 ;最终的差异产物亚克隆到Puc 18载体 ,测序及并进行生物信息学分析 ;半定量RT PCR对筛查到新的基因进行初步的鉴定 .结果发现 9个新ESTs ,2个新基因 .这 2个新基因分别与人及小鼠的丝氨酸蛋白酶抑制因子F ,及真核细胞转录启动因子 3亚单位 5 (EIF 3epsilon)基因有高度的相似性 (>90 % )并在类似普通 2型糖尿病大鼠肾脏组织表达上调 .推测 2个新基因分别是大鼠的丝氨酸蛋白酶抑制因子F及真核细胞转录启动因子 3亚单位 5 .两个新基因在类似普通 2型糖尿病大鼠肾脏组织表达上调 ,可能与类似普通 2型糖尿病大鼠肾脏损害相关 .同时 ,对新基因RS91进行了全长cDNA克隆

Identifying Differences of Gene Expression in Renal Tissue of Type 2 Diabetic Rats by Representational Difference Analysis of cDNA

Representational difference analysis of cDNA(cDNA RDA) was applied to identify differences of gene expression in renal tissue of the type 2 diabetic rats and study the underlying molecular machanism of diabetes and its related complications.The difference bands were generated by cDNA RDA. The final difference products was subcloned into the pUC 18 vector and sequenced. The expressed sequence tags (ESTs) were analysed by bioformatics and the expression levels of ESTs and genes were then verified by semi quantitative RT PCR primitively. 9 novel ESTs and 2 novel genes were obtained in renal tissue of the rat. These novel genes were closely similar to serine (or cysteine) proteinase inhibitor, clade F and eukaryotic translation initiation factor 3, subunit 5 (EIF 3 epsilon) and up regulated in renal tissue of the type 2 diabetic rats. Full length cDNA of a novel gene (RS91) was cloned by in silico cloning. It is presumed that the novel genes may be serine proteinase inhebitor, clade F, and EIF 3 epsilon of rats, respectively and may be related to renal impairment of the type 2 diabetic rat.