小鼠小脑区域性特异表达基因的分离和筛选——乳化酚递减杂交

Screening and Isulatino the Cerebellar Specific Expressed mRNA in Mouse: Subtractive Hybridization by Phenol Emulsion Reassuciation Technique

哺乳类脑组织有着极其复杂和众多的基因表达,许多遗传性神经退行性疾病起源于不同的脑功能区域的基因表达缺陷。分离和筛选小脑特异表达基因是认识小脑疾病的重要途径。本文采用递减杂交法,大脑前皮层和肝组织单链cDNA为驱动物与小脑双链cDNA进行乳化酚杂交,经过重组噬菌体λgt11 DNA连接选择,特异性地克隆那些仅在小脑中优势表达的cDNA。杂交后的小脑eDNA文库容量仅是起始文库容量的0.049%(2.5×10~3/5.15×10~6)。用小脑和肝cDNA探针与PERT cDNA进行克隆杂交和斑点印迹杂交,结果显示绝大多数克隆仅与小脑cDHA杂交阳性。选择10个克隆cDNA与小脑等5种组织mRNA做Northern杂交,有2个小脑特异表达克隆,6个小脑优势表达克隆。对其中PC7和PD8克隆cDNA做部分DNA序列分析,它们是以前未报道过的新基因。

There are extremely complicated and numerous gene expressions in mammalian brains. Each region of the brain contains a set of mRNA expressed only in that region and these mRNA would be related to presumed region of specific function. Genes preferentially expressed in cerebellum (CB) are therfore the reasonable candioate genes for site of genetic lesions in CB neurodegenerative disorders. In this study, the subtractive hybridization was introduced into screening and isolating CB specific mRNA. Testing CB and front cortex (CTX) cDNA libraries were built, which covered most low abundantly expressed mRNA. By applying over 100 fold excessive CTX and liver single strand cDNA to the CB double strand cDNA, sub-tractive hybridization was carried out by phenol-emulsion-reassooiation-technique (PERT), in which the common expressed housekeep genes would be eluted by restrict site ligation, and CB specific cDNA flanking with EcoRI site in both ds cDNA ends could be cloned into lamda gill phage. PERT subtraction reduced CB cDNA libraries from initial 5.15×106 to remaining 2.5 × 108 recombinants, which were most likely CB specific expressed cDNA. Random Twenty re-combinants (over 2 kb inserts) were amplified randomly by polymerase chain reaction (PGR) and were hybridized with CB cDNA and liver cDNA probes in dot blot. 14 inserts positively-hybridized with CB cDNA probe and only 3 inserts showe4 signals with liver cDNA probe. Colony blot presented similar results. 10 cDNA clones which affinited to CB cDNA probe were-selected for the Northern blot. In five tissures, 2 cDNA clones binded only in CB mRNA channal, 6 were proved preferential expression in CB and low abundant expression in 4 other tissues, 1 clone was a housekeep gene aad 1 could not be detected in all tissues. The patially sequencing of clone PC7 and PD8 were introduced into Computer Gene Bank Data Base. No homologous complements were found between these two clones and over ten thousands genes that have been sequenced. The two cloneswere newly reported. According to our experiment, we believe that: 1) CB specific cally expressed mRNAs are much less than expected comparing with other brain regions; 2) most of CB mRNA expressions are in a preferential way other than unique expression.