Development and application of ESS method

我们已经开发了一种方法来制备高质量的消减cDNA文库，该文库允许大规模分离转录诱导的mRNA（cDNA）种类。为了有效地制备这样的消减cDNA文库，我们首先构建了一个载体（pAP 3neo），该载体允许通过新霉素进行选择，通过T7 RNA聚合酶将cDNA插入物转化为RNA形式，通过f1辅助噬菌体产生用于消减的单链cDNA，从cDNA的5 '末端进行快速DNA千测序，并通过SV 40启动子在裂殖酵母和哺乳动物细胞中表达。利用该载体，我们制备了6个实验系统的消减cDNA文库，即小鼠产精子睾丸，剪切应力下的人血管内皮细胞，氮饥饿后减数分裂过程中的裂殖酵母细胞，兔破骨细胞与脾脏的比较，小鼠模拟细胞，以及低和高转移特性的小鼠黑色素瘤细胞。我们还开发了一种逐步消减方法，该方法允许对消减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，我们可以在我们所应用的所有六个实验系统中分离出大量新的cDNA克隆。它们被综合命名为TAU（Transcription in Adult testis Upregulated）、SSR（Shear Stress Responsive）、meu（meotic expression upregulated）、OCS（Osteoclast Specific）、TIM（Transcription increased in mimouse）和TIB（Transcription increased in BL6 mouse）。到目前为止，我们已经检查，其中许多显示生物学上的重要功能。这些结果表明，我们的策略是适用于各种生物学现象，其中转录上调或下调发挥了关键作用。他们对分离的新基因的分析，应该有助于我们理解那些无法解决的生物学问题的调节机制。

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.