Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Manager

Biotechnology and Applied Biochemistry (2001) 33, (99–105) (Printed in Great Britain)

PDF

Legacy HTML

Chromosome localization and gene-copy-number quantification of three random integrations in Chinese-hamster ovary cells and their amplified cell lines using fluorescence in situ hybridization

Julian Davies and Mitchell Reff¹

IDEC Pharmaceuticals Corporation, Molecular Biology Department, 3010 Science Park Road, PO Box 919080, CA 92191–9080, San Diego, U.S.A.

Abbreviations used: CHO, Chinese-hamster ovary; DAPI, 4,6-diamidino-2-phenylindole; DHFR, dihydrofolate reductase; DIG, digoxigenin; FISH, fluorescence in situ hybridization; G418, Geneticin; SFMII, serum-free medium.

¹ To whom correspondence should be addressed (e-mail mreff@idecpharm.com).

We have used fluorescence in situ hybridization (FISH) to localize three random Chinese-hamster ovary (CHO) cell chromosomal integration sites and determine gene copy number in their amplified cell lines. Metaphase FISH showed all three to have integrated into different chromosome positions on different chromosomes. All three Geneticin parent cell lines were found to have single integration sites by Southern-blot analysis, and these data were confirmed using both metaphase and interphase FISH. Following amplification, metaphase FISH showed that amplification in two of the cell lines occurred by duplication in the chromosome arm where the integration occurred. However, for one cell line, amplification resulted in the translocation of amplified genes from one marker chromosome to another. Interphase FISH showed an expected increase in gene copy number upon amplification with methotrexate.

Received 24 November 2000; accepted 17 December 2001

Portland Press Ltd © 2001