Establishment of representative cDNA library from signal cell.

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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.