Development and application of ESS method

我们开发了一种制备高质量消减cDNA文库的方法，该方法允许大规模分离转录诱导的mRNA(CDNAs)物种。为了有效地制备这样的消减文库，我们首先构建了一个载体(PAP3neo)，它可以用新霉素进行选择，用T7RNA聚合酶将插入的cDNA片段转化为RNA形式，用辅助噬菌体产生用于消减的单链cDNAs，从cDNAs的5‘端快速测序，并通过SV40启动子在分裂酵母和哺乳动物细胞中表达。利用该载体构建了小鼠生精睾丸、剪切力作用下的人血管内皮细胞、氮饥饿后的减数分裂酵母细胞、兔破骨细胞与脾的比较、小鼠模拟细胞和小鼠黑色素瘤细胞等6个实验系统的消减文库。我们还开发了一种逐步消减方法，可以对消减后的cDNA文库中的克隆进行有效和全面的分析。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出大量新的cDNA克隆。分别命名为TAU(成人睾丸转录上调)、SSR(剪应力反应)、MEU(减数分裂表达上调)、OCS(破骨细胞特异性)、TIM(模拟上调转录)和TIB(BL6小鼠上调转录)。据我们所知，它们中的许多都具有重要的生物学功能。这些结果表明，我们的策略适用于各种各样的生物现象，其中转录上调或下调起着关键作用。他们对分离的新基因的分析应该有助于我们理解其他无法解决的生物学问题的调控机制。

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.