Development and application of ESS method

ESS方法的开发与应用

基本信息

批准号：
07558216
负责人：
NOJIMA Hiroshi
金额：
$ 3.01万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

We have developed a method to prepare a subtracted cDNA library of high quality that allows the large-scale isolation of transcriptionally induced mRNA (cDNA) species in general. To prepare such subtracted cDNA library efficiently, we first constructed a vector (pAP3neo) that allows selection by neomycin, conversion of the cDNA insert to the RNA form by T7 RNA polymerase, generation of single-stranded cDNA for subtraction by fl helper phage, rapid DNA kilo-sequencing from the 5'ends of cDNAs, and expression in both fission yeast and mammalian cells by the SV40 promoter. Using this vector, we prepared subtracted cDNA libraries of six experimental systems, namely, mouse sperm-producing testis, human angioendothelial cells with shear stress, fission yeast cells during meiosis after nitrogen starvation, rabbit osteoclast cells as compared with spleen, mouse mimutant cells, and mouse melanoma cells of low and high metastatic traits. We also developed a stepwise subtraction method that allows the efficient and comprehensive analysis of the clones in the subtracted cDNA library. By applying these techniques, we could isolate a large number of novel cDNA clones in all of these six experimental systems we have applied. They are comprehensively named as TAU (Transcription in Adult testis Upregulated), SSR (Shear Stress Responsive), meu (meotic expression upregulated), OCS(Osteoclast Specific), TIM (Transcription increased in mimouse) and TIB (Transcription increased in BL6 mouse). So far as we have examined, many of them displayd biologically important functions. These results indicate that our strategy is applicable to a wide variety of biological phenomena in which the transcriptional up or down regulation play the pivotal role. Their analysis of the isolated novel genes should shed light on our understanding of the regulatory mechanisms of otherwise unresolvable biological problems.

我们已经开发了一种方法来制备具有高质量的减去cDNA文库，该文库通常可以大规模分离转录诱导的mRNA（cDNA）物种。 To prepare such subtracted cDNA library efficiently, we first constructed a vector (pAP3neo) that allows selection by neomycin, conversion of the cDNA insert to the RNA form by T7 RNA polymerase, generation of single-stranded cDNA for subtraction by fl helper phage, rapid DNA kilo-sequencing from the 5'ends of cDNAs, and expression in both fission yeast and mammalian cells by the SV40发起人。使用该载体，我们准备了六个实验系统的减去cDNA文库，即，小鼠精子产生睾丸，具有剪切应激的人血管性内皮细胞，氮饥饿后减数分裂，兔骨骨细胞在减数分裂过程中的裂变酵母细胞，与脾，小鼠Mimutant细胞和小鼠Mimutant和小鼠糖浆和低含量相比。我们还开发了一种逐步减法方法，该方法允许对减去cDNA文库中克隆的有效和全面分析。通过应用这些技术，我们可以在我们应用的所有六个实验系统中分离大量新型cDNA克隆。它们被全面命名为tau（成人睾丸的转录上调），SSR（剪切应力响应），MEU（MEU表达上调），OCS（破骨细胞特异性），TIM（MIMOSE中的转录增加）和TIB（转录增加）（在BL6小鼠中的转录增加）。据我们所研究，其中许多显示生物学上重要的功能。这些结果表明，我们的策略适用于多种生物学现象，其中转录向上或向下调节起关键作用。他们对孤立的新基因的分析应阐明我们对原本不可避免的生物学问题的调节机制的理解。