Bacterial artificial chromosome library for genome-wide analysis of Chinese hamster ovary cells

✍ Scribed by Takeshi Omasa; Yihua Cao; Joon Young Park; Yasuhiro Takagi; Shuichi Kimura; Hidenori Yano; Kohsuke Honda; Shuichi Asakawa; Nobuyoshi Shimizu; Hisao Ohtake

• Publisher: John Wiley and Sons
• Year: 2009
• Tongue: English
• Weight: 866 KB
• Volume: 104
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/bit.22463

✦ Synopsis

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.