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

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.

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

“…As indicated above, one of the reasons for the varying results observed in the aforementioned studies is the high heterogeneity of CHO cells, which is in part due to their chromosomal instability and high rate of genomic rearrangements [11][12][13][14]. Two effects contribute: genomic variation between cell lines is observed [5], as well as what has been termed phenotypic drift: the slow but continuous changes and adaptations observed in cells maintained in culture for a prolonged period of time [15,16].…”

Chinese hamster genome sequenced from sorted chromosomes

“…For library construction, we obtained 80-620 ng of DNA for each sorted chromosome and prepared, in addition, a 5,000-bp mate-pair sequencing library from whole genome DNA. We sequenced the libraries on an Illumina (San Diego) Genome Analyzer IIx, using TrueSeq PE Cluster Kit v5-CS-GA and TrueSeq SBS Kit v5-GA and generated ~70-fold genome coverage, assuming a genome size of 2.8 Gb for the Chinese hamster 6 . Subsequently, 1.4 billion reads were assembled into a draft sequence for the separated chromosomes using ALLPATHS-LG 7 .…”

Chromosome identification and its application in Chinese hamster ovary cells

“…The primary Chinese hamster cells were isolated from lung tissue of 4 weeks old female Chinese hamster [7,8]. The structure of the Dhfr amplicon derived from CHO DR1000L-4N cells constructed from CHO DG44 cells was determined previously [7]. The structure has a large palindrome structure containing a small inverted repeat in the junction region.…”

“…Chromosome spreads were prepared from exponential-phase cultures and BAC-FISH to chromosome spreads was carried out as described previously [7]. In brief, the BAC probes were detected using fluorescein isothiocyanate (FITC)-labeled streptavidin or an anti-DIG-rhodamine antibody.…”

“…Two sub clones of CHO cells, proline-requiring CHO K1 [2] and the dihydrofolate reductase (DHFR) gene-deficient CHO DG44 [3], are the most widely used for both scientific research and industrial applications [4][5,6]. Previously, we constructed a genomic bacterial artificial chromosome (BAC) library from mouse Dhfr -amplified CHO DR1000L-4N cell genome, which was provided 5-fold coverage of the CHO cell genome and analyzed the structure of amplicons of exogenous Dhfr amplification [7]. The BAC clones of this library could be landmarks for a physical map for CHO cell genome that are essential to the basic research and industrial application of CHO cell genome.…”