Bacterial artificial chromosome library for genome‐wide analysis of Chinese hamster ovary cells |
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| Authors: | Takeshi Omasa Yihua Cao Joon Young Park Yasuhiro Takagi Shuichi Kimura Hidenori Yano Kohsuke Honda Shuichi Asakawa Nobuyoshi Shimizu Hisao Ohtake |
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| Institution: | 1. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2‐1 Yamadaoka, Suita, Osaka 565‐0871, Japan;2. Department of Aquatic Biosciences, Graduated School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo‐ku, Tokyo, Japan;3. Advanced Research Center for Genome Super Power, Keio University, Tsukuba, Ibaraki, Japan |
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| Abstract: | Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene‐amplified CHO DR1000L‐4N cell line for genome‐wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC‐FISH). Thirteen BAC‐FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR‐deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165‐kb DNA region containing exogenous Dhfr was cloned from the BAC library using high‐density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome. Biotechnol. Bioeng. 2009; 104: 986–994. © 2009 Wiley Periodicals, Inc. |
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| Keywords: | bacterial artificial chromosome library Chinese hamster ovary cells fluorescence in situ hybridization |
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