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 autosomes and 2 sex chromosomes). However, CHO cells have an altered karyotype. In the parental cell line of CHO-K1, 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. 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 ± 0.0088 for the Chinese hamster cells and 0.1630 ± 0.0058 for CHO cells 10. Consequently, the 2C DNA amounts are 5.33 pg and 5.46 pg, respectively. 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 1C-value of 3.12 pg which perfectly fits to the 3.11 ± 0.16 pg measured previously by Greilhuber et al. using Feulgen 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. 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 5.3–5.53 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 1Austrian Centre of Industrial Biotechnology (ACIB GmbH) Muthgasse 11, 1190 Vienna, Austria 2Department 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