1993 Fiscal Year Final Research Report Summary
Isolation of undifferentiated cell-specific genes by the gene trap technique
| Project/Area Number |
04670148
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Kumamoto University |
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Principal Investigator |
SETOYAMA Chiaki Kumamoto University School of Medicine, Lecturer, 医学部, 講師 (60040250)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Cell differentiation / F9 cells / Promoter trap technique / Undifferntiated cell-specific gene / Zinc finger motif / DNA binding protein / Neural crest cell |
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| Research Abstract |
(1)We introduced a promoter trap vector carrying a neo gene as a selectable marker into F9 cells and established four cell lines in which the expression of neo gene is under the control of an endogenous gene that is active only in the undifferentiated F9 cells. Using one of these cell lines, G19, we isolated the undifferentiated cell-specific gene, and named the Zfp-57 gene.
(2)Two different Zfp-57 transcripts (1.8 and 3.2 kb) were identified in the undifferentiated F9 cells, and the levels of these transcripts were decreased significantly within a short time after induction of differentiation. The Zfp-57 cDNAs corresponding to the two RNAs were isolated, and a comparison of the nucleotide sequences revealed that their coding regions were completely identical, but they differed both in length and in sequence of the 3'-untranslated region. The Zfp-57 cDNA encoded a protein consisting of 421 amino acids with an extremely high content of basic amino acid residues and multiple zinc finger motifs.
(3)The Zfp-57 gene showed the restricted patterns of expression in the adult mouse organs and in the particular stage of mouse embryos.
(4)To determine the intracellular localization of the Zfp-57 protein, we raised a polyclonal antibody against a synthetic peptide spanning the residues 121-139 of the Zfp-57 protein. The Zfp-57 antiserum showed striking nuclear staining in undifferentiated F9 cells, whereas most cells showed negative staining in the nucleus after treatment with retinoic acid.
(5)We investigated the expression of the Zfp-57 protein in developing mouse embryo by immunochemical analysis, and found that in the 13-day-embryo the Zfp-57 protein was detected only in the neural crest cell-derived organs.
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