Chromosome identification and its application in Chinese hamster ovary cells
© Cao et al; licensee BioMed Central Ltd. 2011
Published: 22 November 2011
Chinese hamster ovary (CHO) cells  are today a very important host for the commercial-scale production of protein pharmaceuticals. Two sub clones of CHO cells, proline-requiring CHO K1  and the dihydrofolate reductase (DHFR) gene-deficient CHO DG44 , are the most widely used for both scientific research and industrial applications [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 . 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. In this study, we constructed the detail chromosomal physical map of CHO DG44 cell and investigated the chromosome rearrangements among CHO K1, DG44, and primary Chinese hamster cells. Moreover, to determine the effect of the palindrome structure on Dhfr amplification in CHO cells, we constructed three types of expression vectors with or without the junction region of the proposed amplicon and investigated the gene amplification and expression levels in transfected CHO DG44 cells.
Materials and methods
Cell lines, culture conditions, construction of vectors and transfection
CHO DG44, CHO K1 and primary Chinese hamster cells were used in this study. 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 . The structure has a large palindrome structure containing a small inverted repeat in the junction region. This small inverted repeat originates from the integrated vector. On the basis of this junction region, three expression vectors were constructed . The pSV2-dhfr/GFP vector (vector A) was constructed from original vector  and GFP. The pcD-core region (vector B) was constructed from the core region (junction region containing two Dhfr copies and one GFP). The pcD-repeat free core region vector (vector C) was constructed from the repeat free core region (part of the junction region containing one Dhfr and one GFP). Three constructed plasmids were transfected into the CHO DG44 cells. In the Dhfr-amplification step, the transfected cells were cultivated with MTX at various concentrations of 50, 100, 250 and 500 nM.
Fluorescence in situ hybridization using BAC clones as hybridization probes (BAC-FISH) and construction of CHO physical map
Chromosome spreads were prepared from exponential-phase cultures and BAC-FISH to chromosome spreads was carried out as described previously . In brief, the BAC probes were detected using fluorescein isothiocyanate (FITC)-labeled streptavidin or an anti-DIG-rhodamine antibody. Chromosomes were counterstained with 4,6,-diamidino-2-phenylindole (DAPI) and observed under an Axioskop 2 fluorescence microscope. Photographs were taken with a CCD camera. After image processing was performed, the ImageJ software (http://rsbweb.nih.gov/ij/) was used to analyze the chromosomal loci of the BAC clone probes and the positions of the centromere on the chromosomes, and expressed as FLpter values .
Results and discussion
Construction of BAC-based physical map for Chinese hamster ovary cells
FISH analysis of gene-amplified chromosomal region of transfected cells 
Ratios of amplified genes located at same position of BAC clone Cg0160E04 on chromosome O.
MTX concentration (nM)
Vector A (%)
Vector B (%)
Vector C (%)
0 (0/14) a
0 (0/11) a
0 (0/15) a
0 (0/10) a
33.3 (4/12) a
18.2 (2/11) a
0 (0/10) a
61.5 (8/13) a
57.1 (8/14) a
0 (0/10) a
70.8 (17/24) a, b
66.7 (6/9) a
In summary, we constructed a BAC-based physical map for CHO DG44 cells and analyzed genome-wide rearrangements of chromosome among CHO cells. This BAC-based physical map will greatly facilitate the studies of CHO cell genome. The BAC clones comprising this physical map could also provide a genome-wide resource for analysis of chromosome rearrangements, chromosome structure, comparative genome hybridization, gene targeting, and functional genomics.
The present work is partially supported by grants from the NEDO of Japan, the Program for the Promotion of Fundamental Studies in Health Sciences of the NIBIO, and the Grant-in-Aid for Scientific Research of the JSPS. CHO BAC library was constructed under the collaboration with Professor S. Asakawa at the University of Tokyo and Professor N. Shimizu at Keio University.
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