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1994 Fiscal Year Final Research Report Summary

Establishment of representative cDNA library from signal cell.

Research Project

Project/Area Number 04557021
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Experimental pathology
Research InstitutionChiba University

Principal Investigator

TANIGUCHI Masaru  School of Med.Chiba Univ.Prof., 医学部, 教授 (80110310)

Co-Investigator(Kenkyū-buntansha) KANNO Masamoto  School of Med.Chiba Univ.Assoc.Prof., 医学部, 助教授 (40161393)
Project Period (FY) 1992 – 1994
Keywordssingle cell / subtraction / Anchor PCR / cDNA Library
Research Abstract

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.

  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Sumida, T.: "Prevention of isulitis and diabetes in β2-microglobulin-deficient non-obese diabetic mice." Int. Immunol.6. 1445-1449 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Makino, Y.: "Extrathymic differentiation of a T cell bearing invariant Vα14Jα281 TCR." Intern. Rev. Immunol.11. 31-46 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sydora, B. C.: "Intestinal intraepithelial lymphocytes preferentially repopulate the intestinal epitheljum." Int. Immunol.5. 743-751 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Makino, Y.: "Extrathymic development of Vα14 positivet cells." J. Exp. Med.177. 1339-1408 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kimoto, H.: "The fetal thymus stores immature hemopoietic cells constituting the thymus stromal element." Int. Immunol.5. 1535-1540 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shirasawa, T.: "Heavy chain variable (V_H) region diversity generated by V_H gene replacement in the progeny of a signal precursor cell transformed with a temperature-sensitive mutant of abelson murine leukemia virus." J. Exp. Med.176. 1209-1214 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Inaba, T.: "Doble-step and inverse polymerase chain reaction for sensitive detection and cloning of T cell receptor variable region sequences." Int. Immunol.3. 1053-1057 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Sumida, T.: "Molecular Approaches to the Study and Treatment of Human Diseases." Yoshida, T. O. and Wilson, J. M., 455 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Makino, Y., Koseki, H., Adachi, Y., Akasaka, T., Tsuchida, K.and Taniguchi, M.: "Extrathymic differentiation of a T cell bearing invariant Valpha14 Jalpha281 TCR." Intern, Rev.Immunol.11. 31-46 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Sydora, B.C., Habu, S.and Taniguchi, M.: "Intestinal intraepitehlial lymphocytes preferentally repopulate the intestinal epithelium." Int.Immunol.5. 743-751 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Makino, Y., Yamagata, N., Sasho, T., Adachi, Y., Kanno, R., Koseki, H., Kanno, M.and Taniguchi, M.: "Extrathymic development of Valpha14 positive T cells." J.Exp.Med.177. 1399-1408 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kimoto, H., Kitamura, K., Sudo, T., Suda, T., Ogawa, Y., Kitagawa, H., Taniguchi, M.and Takemori, T.: "The fetal thymus stores immature hemopoietic cells capable element." Int.Immunol.5. 1535-1540 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Shirasawa, T., Miyazoe, I., Hagiwara, S., Kimoto, H., Shigemoto, K., Taniguchi, M.and Takemori, T.: "Heavy chain variable (V^H) region diversity generated by V^H gene replacement in the progeny of a single precursor cell transformed with a temperature-sensitive mutant of abelson murine leukemia virus." J.Exp.Med.176. 1209-1214 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Inaba, T., Koseki, H., Suzuki, M.and Taniguchi, M.: "Double-step and inverse polymerase chain reaction for sensitive detection and cloning of T cell receptor variable region sequences." Int.Immunol.3. 1053-1057 (1991)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1996-04-15  

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