Establishment of representative cDNA library from signal cell.

虽然基因表达模式的变化是现代生物学的主要研究热点，但分化系统的异质性经常构成一个困难的技术挑战。在这个项目中，我们开发了一种简单的方法来同时扩增单个细胞中存在的大多数多腺苷化mRNAs的序列，克服了以前方法的局限性。(1)所有步骤均在原悬浮液中进行，不需要提取或沉淀步骤。因此，该程序可以可靠地应用于多个样品，每个样品都小到一个电池。(2)采用“细胞内聚合酶链式反应”技术，无需细胞裂解步骤即可进行基因合成和聚合酶链式反应。(3)最初的cDNA转录本的大小被限制在随后扩增最有效的范围内。结果是扩增产物，它在特定序列的存在和相对丰度方面代表了起始样本。(4)通过生物素化的引物和链霉亲和素的差减，我们成功地扩增了大约10倍，目前我们仍有一个突破点。首先，从杂交瘤或体内培养的单个细胞中成功地扩增出cDNAs，然后检测到扩增出的cDNA。然而，我们需要50个细胞来扩增和检测来自正常细胞(淋巴细胞或胸腺细胞等)的低拷贝数的cDNA(例如转录因子或膜蛋白)。其次，由于两端都有一些Adaptor-素数的存在，用这种减法不能丰富十倍以上的内容。我们现在正在努力解决这些问题。我们也开始应用这种方法来鉴定造血干细胞的承诺因子，这些干细胞可以从单个细胞重建所有的造血系统。

While the changing patterns of gene expression that underlie differentiation are major focus of interest in modern biology, the heterogeneity of differentiating systems frequently poses a difficult technical challenge.In this project, we developed a simple procedure for simultaneous amplification of sequences representing the majority of polyadenylated mRNAs present in a single cell which overcomes the limitations of the earlier methods. (1) All steps are performed in the original suspension without extraction or precipitation steps. As a result, the procedure can be applied reliably to multiple samples each as small as a single cell. (2) cDNA synthesis and PCR reactions are performed without cell-lysis step applied from "In-cell PCR" technique. (3) The initial cDNA transcripts are limited in size to a range in which subsequent amplificaiton is maximally efficient. The resuls is an amplified product which is representative of the starting sample in terms of presence as well as relative abundance of specific sequences. (4) , By subtraction with biotinylated primer and streptavidine, we succeeded about ten-fold enrichment.At present time, we still have a point to break-through. Firstly, we succeeded cDNA amplification from a single cell derived from hybridoma or in vivo cultured cell line and then detected amplified cDNAs. However, we need 50 cells to amplify and detect low copy number of cDNAs (e.g.transcription factors or membrane proteins)starting from normal cells(lymphnode cell or thymocyte, etc.) . Secondly we can not enrich more than ten times by this subtraction method due to the presence of some Adaptor-prime at both ends. We are now trying to solve these problems. We also start applying this method to identify committment factors for the hematopoietic stem cells which can reconstitute all heomatopoietic system from a single cell.