用mRNA差异显示法分离大鼠吗啡依赖相关基因

A preliminary study on the isolation of specially expressed genes of morphine dependent rat by differential display PCR

目的：分离并克隆大鼠吗啡依赖相关基因。方法：ＳＤ大鼠用递增剂量的吗啡作皮下注射，形成吗啡依赖的动物模型。分离鼠脑的中脑导水管周围灰质、伏核、纹状体等核团，提取总ＲＮＡ，用ｍＲＮＡ差异显示（ｄｉｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙＰＣＲ，ＤＤＰＣＲ）的方法，筛选吗啡依赖大鼠特异表达产物的ｃＤＮＡ片段。用３组锚定引物（Ｔ１２ＭＮ，Ｍ＝Ａ，Ｇ，Ｔ或Ｃ，Ｎ＝Ａ，Ｃ或Ｇ）和６组随机引物（ＡＰ１～ＡＰ６）作不同组合进行ＰＣＲ扩增。结果：获得了８０余个差异表达产物的ｃＤＮＡ片段，经斑点杂交初步筛选，克隆了１０个阳性片段，选取４个克隆进行序列分析，获得了４个ｃＤＮＡ片段。结论：通过ＢＬＡＳＴ数据库序列比较，现已确认获得了４个吗啡依赖表达的新基因片段。

Objective: To isolate and clone morphine dependent genes of SD rat. Methods: SD rats were used for the morphine dependent model. They were injected with increasing dose of morphine for 10 days. At single inject, the dose of morphine was from 5 mg/kg to 120 mg/kg. The rats were then scarified and the brain region was disassociated. The nucleus such as periaqueductal gray (PAG), nucleus accumbens, striatum were selected for total RNA extraction. Differential display PCR (DD PCR) of mRNA was used for isolating the specific expressed genes. Three sets of anchor primers (T 12 MN, M=A,G,T or C; N=A,C or G) and 6 sets of arbitrary primers(AP1～AP6) were used for DDPCR amplification. By combination of anchor primers and arbitrary primers, series of PCR reaction were preformed. Results: More than 80 cDNA fragments, which might represent the differentially expressed genes, were obtained by DDPCR method. Dot blot was used for further selection of positive cDNA, and more than 10 cDNA fragments with strong positive signals were cloned. Four clones were sequenced. Four cDNA fragments were obtained. Conclusion: Based on the sequence homologous comparison with NIH BLAST data, four specially expressed new gene fragments have been isolated and cloned by using mRNA differential display PCR (DDPCR) method.