Functional analysis of B cell receptors utilizing

Research Project

Project/Area Number	06670362
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Immunology
Research Institution	University of Tokyo (1995) Juntendo University (1994)
Principal Investigator	NAKAMURA Tetsuya Assistant Professor, 医学部(病), 助手 (30189047)
Project Period (FY)	1994 – 1995
Project Status	Completed (Fiscal Year 1995)
Budget Amount *help	¥2,100,000 (Direct Cost: ¥2,100,000) Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000) Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Keywords	B lymphocyte / antigen receptor / Ig-alpha / Ig-beta / B細胞リセプター
Research Abstract	(1) I accomplished genomic cloning of the human Ig-beta gene, and revealed that Ig-beta was encoded by four exons spanning over approximately 2 kilo bases. This information together with the previous result of the genomic sequence of IG-alpha provide us informations necessary for gene targeting of Ig-alpha and Ig-beta. However, preliminary experiments have suggested that the efficiency for gene transfection into B-lineage cells is so poor that it seems difficult to establish Ig-alpha or Ig-beta-deficient cells by this methods. (2) As an alternative method, I tried to obtain variant B cell lines which lack Ig-alpha or Ig-beta expression. For this purpose, Ramos and Daudi B cells were given with 300-500 rad of irradiation to induce mutation, and then cultured in the medium containing anti-IgM antibody which suppress growth of surface IgM-positive B cells. After several weeks of cultures, IgM-negative B cells were obtained by excluding IgM-positive cells which adhere the plastic dishes coated with anti-IgM antibody. (3) The IgM-negative B cells were cloned by limiting dilution, and the clones which lack Ig-alpha and Ig-beta expression are now being selected. Introduction of mutated Ig-alpha or Ig-beta gene into these clones will enable us to attain the initial purpose. (4) While I have analyzed the genomic sequence of human Ig-beta, I found the alternatively spliced Ig-alpha and Ig-beta transcripts. These transcripts were thought to have relation to down-modulation of B cell receptors in B cell differentiation.