Development and application of ESS method

点击翻译按钮获取中文摘要

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.