1998 Fiscal Year Final Research Report Summary
Isolation of new genes related to hematopoietic cells using gene trap screening in ES cells
| Project/Area Number |
09670349
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Immunology
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| Research Institution | The University of Tokyo (1998)
Research Institute, Osaka Medical Center for Maternal and Child Health (1997) |
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Principal Investigator |
YOSHIDA Nobuaki The University of Tokyo, The Institute for Medical Science, Professor, 医科学研究所, 教授 (10250341)
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| Project Period (FY) |
1997 – 1998
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| Keywords | gene trap / ES cell / hematopoietic cell / growth factor / cell differentiation / cloning |
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| Research Abstract |
We constructed a gene trap vector contained a En-2 splice acceptor site, IRES signal, the LacZ gene as a reporter gene and neomycin resistant gene under the control of an actin promoter. This vector works effectively by using retinoic acid as a factor for ES cell differentiation in vitro . We introduced this vector into D3 or E14-1 ES cells and established several thousands of clones. We cultured these clones on OP-9 feeder layers derived from op/op mice and stained with X-gal at day 8 of culture. Although very few LacZ positive clones were detected, we picked up these cells and made cDNA library from these positive clones. We sequenced more than 10 cDNA clones and identified some unknown gene fragment. Most of them were ubiquitously expressed in adult mouse tissues revealed by Northern blot analysis but a few showed tissue specific expression to some extent. But none of these clones were expressed exclusively in hematopoietic tissues.
ES cells differentiate into different tissues in vitro, but these are heterogeneous. Therefore, we are trying to fish out culture conditions which support ES cell differentiate to specific lineage in vitro or sorting out differentiated cells using lineage specific surface markers by FACS.ES cells established from other species can be used in this gene trap approach in the future.
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