Screening of Novel Epilepsy-Related Genes and Isolation and Identification of cDNAs

Summary: Twenty cDNA differential fragments were isolated from the hippocampus of rats in epileptic state. using mRNA differential display technique. Four fragments were sequenced and compared with the known sequences in the Genebank, which showed that ERGS, ERG12, ERG12 had no significant identity to any known sequences; ERG14 had 64 %-69 % identity to micro- tubulin-associated protein of the rat. Because the differential expression of these genes was caused by epilepsy inducer coriaria lactone (CL) and anti-epilepsy drug MK-801 and ERGS might be a novel candidate epilepsy gene; ERG11 and ERG12 might be novel candidate anti-epilepsy genes. Since the microtubulin-associated protein is closely associated with the collateral sprouting of mossy.fibers in the hippocampus of seizured rat, the high expression of ERG14 in the early stage of epilepsy might predict the growth of axon and formation of synapse.

mRNA差别显示法在大鼠肾炎相关基因筛选和克隆中的应用

以系膜增殖性肾炎大鼠肾脏皮质为材料，对ｍＲＮＡ差别显示法进行了方法学的探索和改进。结果发现，影响实验结果的主要因素为引物浓度、退火温度、模板投放量等，并摸索出最佳反应参数，使每次反应可稳定地显示８０～１００个条带。用该方法初步获得部分基因片断，经克隆测序并与ＧｅｎｅＢａｎｋ比较证实为新基因。证明该方法是一个操作简单、实验周期短、重复性好、费用较低的有效的筛选和克隆基因的好方法，具有良好的发展前景。

利用反向mRNA斑点印迹从抑制消减杂交阳性克隆中筛选差异表达基因

目的 :建立一种从抑制消减杂交阳性克隆中筛选差异表达基因的方法。方法 :以阵列方式将抑制消减杂交阳性克隆的PCR产物点加于尼龙膜上 ,以标记 polyA+ RNA为探针进行反向杂交 ,化学发光法检测 ,在图象分析仪上进行光密度扫描 ,计量平均单位面积光密度 ,扣除背景 ,计算两个阵列各对应点之间光密度值的比值R(ratio)。差异表达基因的阳性判定标准为R值大于或等于 2 .0 0。结果 :通过反向mRNA斑点印迹技术可以从抑制消减杂交阳性克隆中把差异表达基因有效地筛选出来。结论 :反向mRNA斑点印迹技术是一种筛选抑制消减杂交差异表达基因的有效方法。