应用银染mRNA差异显示法寻找高温致神经管畸形差异表达基因

目的 :寻找高温致神经管畸形的差异表达基因。方法 :在高温致神经管畸形的动物模型上 ,分别于高温处理后 2 4、48和 72小时 ,提取鼠胚神经管组织总RNA和正常对照组鼠胚相应时间的神经管组织总RNA ,反转录合成cDNA第一链后进行差异显示PCR扩增 ,采用PAGE和银染技术显示差异条带 ,回收差异条带并经PCR二次扩增后 ,用点杂交方法筛除假阳性条带 ,再用Northern印迹杂交进一步鉴定。结果 :在高温致畸的鼠胚神经管组织中筛选到一个特别明显的差示cDNA片段N3 2 ,该片段所在基因在高温致畸的胚胎神经管组织中的表达远低于正常同龄胚的神经管组织。结论 :N3 2片段所在基因的低表达与高温致神经管畸形相关。

Identification of genes related to hyperthermia induced neural tube defects by mRNA differential display method

Objective:To identify the genes related to hyperthermia induced neural tube defects. Methods: Total RNAs of neural tube tissues were extracted from hyperthermia treated golden hamster embryos at 24 h, 48 h and 72 h after hyperthermia treatment. Total RNAs of normal control group were extracted correspondingly. Differences in gene expression between the 2 groups were investigated by an improved protocol of mRNA differential display with silver staining: a highly sensitive staining without radioactivity. Results: Obvious differences in gene expression were observed between hyperthermia treated group and normal control group. After dot blot and Northern blot analysis, 1 cDNA fragment named N32 was confirmed to express much stronger in neural tube tissue of normal group than in that of hyperthermia treated group. Conclusion: The lower expression of N32 gene is related to the neural tube defects induced by hyperthermia.