Communication to the Editor

Whole Genome Sequencing Improves

Estimation of Nuclear DNA Content of
Chinese Hamster Ovary Cells
CHINESE hamster ovary (CHO) cells are the most widely
used mammalian host for the expression of recombinant proteins for therapeutic application. The generation of stable
CHO production cell lines as well as the methods for characterization and validation are well established. High-expressing
cell lines are generally produced by exploiting the phenomenon of gene amplification which is widespread in eukaryotic
cells (1). The host cells (e.g., dihydrofolate reductase-deficient
CHO cells) are transfected with an expression vector containing an adequate amplification marker (e.g., dihydrofolate
reductase) and the gene of interest. Applying increasing
amounts of the appropriate inhibitor (e.g., methotrexate)
leads to the amplification of the amplification marker as well
as genetically linked sequences including the gene of interest
(Fig. 1). In order to study the efficiency of the applied amplification strategy, the correlation between transgene copy number and target transcript or target protein levels, or the
stability of the copy number over cultivation time, it is essential to know the number of target genes that are integrated in
the genome. The method most frequently used to determine
gene copy numbers is quantitative real-time PCR (qPCR).
The calculation of the absolute gene copy number per cell
requires the knowledge of the number of genome copies in
the template used for amplification. However, the assumed
nuclear DNA content of CHO cells varies considerably
between different studies ranging from 3.3 pg (2) to 6.0 pg per
cell (3) or is not mentioned at all in many other publications.
Although the sequence of the CHO-K1 cell line became available recently (4), there is still some uncertainty among scientists about the nuclear DNA amount in unreplicated nuclei of
CHO cells. The diploid genome of a normal Chinese hamster
(Cricetulus griseus) cell has 22 chromosomes (10 pairs of

Received 5 April 2013; Revised 14 May 2013; Accepted 7 June 2013

Grant sponsors: Federal Ministry of Economy, Family and Youth
(BMWFJ), Federal Ministry of Traffic, Innovation and Technology
(bmvit), Styrian Business Promotion Agency SFG, Standortagentur
Tirol and ZITTechnology Agency of the City of Vienna through the
COMET-Funding Program managed by the Austrian Research Promotion Agency FFG.

autosomes and 2 sex chromosomes). However, CHO cells

have an altered karyotype. In the parental cell line of CHOK1, only 8 of the determined 21 chromosomes appear to be
normal as compared to the euploid Chinese hamster chromosomes and the remaining 13 chromosomes (Z group) contain
translocations, deletions, and pericentric inversions (5).
Nevertheless, apart from the X2 chromosome all of the
euploid chromatin is present in CHO cells (5). Consequently,
CHO cells have a nuclear DNA content close to that of normal
diploid Chinese hamster cells.
Furthermore, the definition of genome size often leads to
confusion. The correct usage of the term genome size and
also the commonly used term C-value has been discussed
extensively in literature (6). For diploid cells, genome size and
C-value are interchangeable and are generally defined as the
amount of nuclear DNA within a haploid (1n) set of chromosomes (7,8). Consequently, the nuclear DNA content of a diploid cell is twice the genome size or the 2C-value. However,
what does this mean for CHO cells? Here, the unreplicated
nuclear DNA content of CHO cells (2C DNA amount) was
estimated in three different ways based on the currently available data from literature providing very similar results.

THE GENOME SEQUENCE OF THE CHO-K1 CELL LINE

In 2011, the genome sequence of the CHO-K1 cell line
was published (4). The assembly contained 2.45 Gb of
genomic sequence and the authors of this study estimated a
genome size of 2.6 Gb (4). As the total amount of chromatin
in CHO cells is close to that in normal Chinese hamster cells
(5), it can be assumed that most of the genomic sequence is
also present twice in CHO cells like it would be in normal

*Correspondence to: Andreas Maccani, Austrian Centre of Industrial

Biotechnology (ACIB GmbH), Muthgasse 11, 1190 Vienna, Austria.
E-mail: andreas.maccani@acib.at
Published online 10 July 2013 in Wiley Online Library
(wileyonlinelibrary.com)
DOI: 10.1002/cyto.a.22331
C 2013 International Society for Advancement of Cytometry
V

Cytometry Part A  83A: 893895, 2013

Communication to the Editor

Figure 1. General procedure of gene amplification to generate stable, high-expressing recombinant mammalian cell lines. [Color figure
can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

diploid cells. Consequently, the 2C genome size is 5.2 Gb.

Applying Eq. (1) which was determined by Dolezel et al. (9)
leads to a 2C DNA amount of 5.3 pg.


DNA contentpg 5genome size bp = 0:9783109
(1)

FEULGEN CYTOPHOTOMETRY
In a study from 1983, Feulgen cytophotometry was used
to determine the nuclear DNA content of various animal cells
relative to the plant Allium cepa (10). Allium cepa is frequently
used as reference for DNA measurements having a 2C-value
of 33.5 pg (11). Although this value originates from a study
published in 1965, it is still the generally accepted estimate
today (7). Greilhuber et al. measured 134 nuclei of mitotic
Chinese hamster lung fibroblasts and 50 nuclei of mitotic
CHO cells which were considered as diploid (2n). The determined DNA amount relative to Allium cepa was 0.1590 6
0.0088 for the Chinese hamster cells and 0.1630 6 0.0058 for
CHO cells (10). Consequently, the 2C DNA amounts are 5.33
pg and 5.46 pg, respectively.

COMPARISON TO THE HUMAN GENOME SIZE

The overall human genome (22 autosomes, X and Y chromosome) was estimated to be 3.08 Gb (12). Subtracting the
size of the Y chromosome leads to a genome size of 3.05 Gb for
a female human cell. Kato et al. measured the genome size of
34 species relative to diploid human fibroblasts using Feulgen
cytophotometry (13). They determined the genome size of the
Chinese hamster to be 91.5% of the female human genome.
Hence, the genome size of the Chinese hamster is 2.79 Gb. As
CHO cells contain 3% less total chromatin than diploid Chinese hamster cells (5), the 2C genome size of CHO cells is 5.41
Gb. Applying Eq. (1) leads to a 2C DNA amount of 5.53 pg.
The human genome size of 3.05 Gb corresponds to a 1Cvalue of 3.12 pg which perfectly fits to the 3.11 6 0.16 pg
measured previously by Greilhuber et al. using Feulgen
894

cytophotometry (10). They determined the nuclear DNA content of CHO cells to be 88% of human, which would lead to a
2C DNA amount of 5.49 pg.

AVERAGE NUCLEAR DNA CONTENT OF CHO CELLS

All considerations were made based on data from the
CHO-K1 cell line or its parental cell line. CHO cells are genetically very unstable leading to considerable genomic heterogeneity between different cells lines (14). For example, the cell
line DG44 has only 20 chromosomes of which only seven are
normal, four can be assigned to the Z group, and the remaining nine are altered chromosomes (15). Consequently, it is
very likely that there are also slight differences in genome size
between cell lines. However, it can be assumed that the variations in the total amount of nuclear DNA are marginal. The
determined 2C DNA amounts of CHO cells were within a narrow range of 5.35.53 pg. For this reason 5.4 pg seems to be a
good estimation of the unreplicated nuclear DNA content of
CHO cells.
Andreas Maccani*
Austrian Centre of Industrial Biotechnology
(ACIB GmbH)
Muthgasse 11, 1190 Vienna, Austria
Wolfgang Ernst1,2
Austrian Centre of Industrial Biotechnology
(ACIB GmbH)
Muthgasse 11, 1190 Vienna, Austria

Department of Biotechnology
University of Natural Resources and Life Sciences
Muthgasse 18, 1190 Vienna, Austria
Reingard Grabherr
Department of Biotechnology
University of Natural Resources and Life Sciences
Muthgasse 18, 1190 Vienna, Austria
Communication to the Editor

Communication to the Editor

LITERATURE CITED
1. Omasa T. Gene amplification and its application in cell and tissue engineering. J Biosci Bioeng 2002;94:600605.
2. Reisinger H, Steinfellner W, Stern B, Katinger H, Kunert R. The absence of effect of
gene copy number and mRNA level on the amount of mAb secretion from mammalian cells. Appl Microbiol Biotechnol 2008;81:701710.
3. Osterlehner A, Simmeth S, Gopfert U. Promoter methylation and transgene copy
numbers predict unstable protein production in recombinant Chinese hamster ovary
cell lines. Biotechnol Bioeng 2011;108:26702681.
4. Xu X, Nagarajan H, Lewis NE, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W,
Hammond S, et al. The genomic sequence of the Chinese hamster ovary (CHO)-K1
cell line. Nat Biotechnol 2011;29:735741.
5. Deaven LL, Petersen DF. The chromosomes of CHO, an aneuploid Chinese hamster
cell line: G-band, C-band, and autoradiographic analyses. Chromosoma 1973;41:
129144.
6. Greilhuber J, Dolezel J, Lysak MA, Bennett MD. The origin, evolution and proposed
stabilization of the terms genome size and C-value to describe nuclear DNA contents. Ann Bot 2005;95:255260.
7. Dolezel J, Greilhuber J. Nuclear genome size: Are we getting closer? Cytometry A
2010;77A:635642.

Cytometry Part A  83A: 893895, 2013

8. Gregory TR. Coincidence, coevolution, or causation? DNA content, cell size, and the
C-value enigma. Biol Rev Camb Philos Soc 2001;76:65101.
9. Dolezel J, Bartos J, Voglmayr H, Greilhuber J. Nuclear DNA content and genome size
of trout and human. Cytometry A 2003;51A:127128.
10. Greilhuber J, Volleth M, Loidl J. Genome size of man and animals relative to the
plant Allium cepa. Can J Genet Cytol 1983;25:554560.
11. Vant Hof J. Relationships between mitotic cycle duration, S period duration and the
average rate of DNA synthesis in the root meristem cells of several plants. Exp Cell
Res 1965;39:4858.
12. International Human Genome Sequencing C. Finishing the euchromatic sequence of
the human genome. Nature 2004;431:931945.
13. Kato H, Harada M, Tsuchiya K, Moriwaki K. Absence of correlation between DNA
repair in ultraviolet irradiated mammalian cells and life span of the donor species.
Jpn J Genetics 1980;55:99108.
14. Cao Y, Kimura S, Itoi T, Honda K, Ohtake H, Omasa T. Construction of BAC-based
physical map and analysis of chromosome rearrangement in Chinese hamster ovary
cell lines. Biotechnol Bioeng 2012;109:13571367.
15. Derouazi M, Martinet D, Besuchet Schmutz N, Flaction R, Wicht M, Bertschinger
M, Hacker DL, Beckmann JS, Wurm FM. Genetic characterization of CHO production host DG44 and derivative recombinant cell lines. Biochem Biophys Res Commun 2006;340:10691077.

895