Abstract
Normal human fibroblasts (MRC‐5 or NTI‐4) were transfected with pSV2‐neo plasmid DNA. Fifty G418‐resistant fibroblast clones were isolated and independently fused to mouse A9 cells. The cell hybrids were selected and isolated in the medium containing G418 plus ouabaln. Since micronuclei were more efficiently induced in these hybrids compared to parental human fibroblasts by colcemid treatment, the transfer of neo‐tagged human chromosomes in the hybrids to mouse A9 cells was performed via microcell fusion. Two hundred A9 microcell hybrids were isolated and karyotyped. Among them, thirteen microcell clones, each containing a single human chromosome 1, 2, 5, 6, 7, 8, 10,11,12,15,18,19 or 20 were established. Isozyme analyses confirmed the presence of each human chromosome in these A9 microcell clones. The results of Southern blot and chromosomal in situ hybridization analyses indicate that the human chromosomes in these clones were tagged with pSV2‐ neo plasmid DNA.
Keywords: pSV2‐neo gene, Chromosome transfer, Microcell fusion, Human mono‐chromosomal library
Full Text
The Full Text of this article is available as a PDF (699.5 KB).
REFERENCES
- 1. ) Koi , M. , Morita , H. , Shimizu , M , and Oshimura , M.Construction of mouse A9 clones containing a single human chromosome (X/autosome translocation) via micro‐cell fusion . Jpn. J. Cancer Res. , 80 , 122 – 125 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. ) Saxon , P. J. , Srivatsan , E. S. and Stanbridge , E. J.Introduction of human chromosome 11 via microcell transfer controls tumorigenic expression of HeLa cells . EMBO J. , 15 , 3461 – 3466 ( 1986. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. ) Weissman , B. E. , Saxon , P. J. , Pasquale , S. R. , Jones , G. R. , Geiser , A. G. and Stanbridge , E. J.Introduction of a normal human chromosome 11 into Wilms' tumor cell line controls its tumorigenic expression . Science , 236 , 175 – 180 ( 1987. ). [DOI] [PubMed] [Google Scholar]
- 4. ) Saxon , P. J. , Srivatsan , E. S. , Leipzig , G. V. , Sameshima , J. H. and Stanbridge , E. J.Selective transfer of individual human chromosomes to recipient cells . Mol. Cell Biol. , 5 , 140 – 146 ( 1985. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. ) Athwal , R. S. , Smarsh , M. , Searle , B. and Deo , S. S.Integration of a dominant selectable marker into human chromosomes and transfer of marked chromosomes to mouse cells by microcell fusion . Somatic Cell Mol, Genet , 11 , 177 – 187 ( 1985. ). [DOI] [PubMed] [Google Scholar]
- 6. ) Lugo , T. G. , Handelin , B. , Killary , A. M. , Housman , D. E. and Fournier , R. E. K.Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes . Mol Cell Biol. , 17 , 2814 – 2820 ( 1987. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. ) Lester , S. C. , LeVan , S. K. , Steglich , C. and DeMars , R.Expression of human genes for adenine phosphoribosyl‐ transferase and hypoxanthine‐guanine phosphoribosyl‐ transferase after genetic transformation of mouse cells with purified human DNA . Somatic Cell Genet. , 6 , 241 – 259 ( 1980. ). [DOI] [PubMed] [Google Scholar]
- 8. ) Koi , M. and Barrett , J. C.Loss of tumor‐suppressive function during chemically induced neoplastic progression of Syrian hamster embryo cells . Proc. Natl. Acad. Sci. USA , 83 , 5992 – 5996 ( 1986. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. ) Shay , J. W. and Clark , M. A.Nuclear control of tumorigenicity in cells reconstructed by PEG‐induced fusion of cell fragments . J. Supramol. Struc. , 11 , 33 – 49 ( 1979. ). [DOI] [PubMed] [Google Scholar]
- 10. ) Yoshida , M. C. , Ikeuchi , T. and Sasaki , M.Differential staining of parental chromosomes in interspecific cell hybrids with a combined quinacrine and 33258 Hoechst technique . Proc. Jpn. Acad. , 51 , 184 – 187 ( 1975. ). [Google Scholar]
- 11. ) Southern , E. M.Detection of sequences among DNA fragments separated by gel electrophoresis . J. Mol Biol. , 98 , 503 – 517 ( 1975. ). [DOI] [PubMed] [Google Scholar]
- 12. ) Harper , M. E. A. , Ulrich , A. and Saunders , G. F.Localization of the human insulin gene to the distal end of the short arm of chromosome 11 . Proc. Natl. Acad. Sci. USA , 78 , 4458 – 4460 ( 1981. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. ) Koi , M. , Morita , H. , Yamada , H. , Satoh , H. , Barrett , J. C. and Oshimura , M.Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa . Mol. Carcinog. , in press . [DOI] [PubMed] [Google Scholar]