Microevolutionary Dynamics of EccDNA in Chinese Hamster Ovary Cells Grown in Fed-batch Cultures Under Control and Lactate-stressed Conditions

Overview

Journal Sci Rep

Specialty Science

Date 2023 Jan 21

PMID 36681715

Authors

Dylan G Chitwood

Qinghua Wang

Stephanie R Klaubert

Kiana Green

Cathy H Wu

Sarah W Harcum

Christopher A Saski

Affiliations

Soon will be listed here.

Abstract

Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.

Citing Articles

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Fu W, MacGregor D, Comont D, Saski C Genes (Basel). 2023; 14(10).

PMID: 37895254 PMC: 10606437. DOI: 10.3390/genes14101905.

Dynamics of Amino Acid Metabolism, Gene Expression, and Circulomics in a Recombinant Chinese Hamster Ovary Cell Line Adapted to Moderate and High Levels of Extracellular Lactate.

Chitwood D, Uy L, Fu W, Klaubert S, Harcum S, Saski C Genes (Basel). 2023; 14(8).

PMID: 37628627 PMC: 10454118. DOI: 10.3390/genes14081576.

References

Verhaak R, Bafna V, Mischel P . Extrachromosomal oncogene amplification in tumour pathogenesis and evolution. Nat Rev Cancer. 2019; 19(5):283-288. PMC: 7168519. DOI: 10.1038/s41568-019-0128-6. View

de Nadal E, Ammerer G, Posas F . Controlling gene expression in response to stress. Nat Rev Genet. 2011; 12(12):833-45. DOI: 10.1038/nrg3055. View

Robinson J, Thorvaldsdottir H, Wenger A, Zehir A, Mesirov J . Variant Review with the Integrative Genomics Viewer. Cancer Res. 2017; 77(21):e31-e34. PMC: 5678989. DOI: 10.1158/0008-5472.CAN-17-0337. View

Yona A, Bloom-Ackermann Z, Frumkin I, Hanson-Smith V, Charpak-Amikam Y, Feng Q . tRNA genes rapidly change in evolution to meet novel translational demands. Elife. 2013; 2:e01339. PMC: 3868979. DOI: 10.7554/eLife.01339. View

Cantarel B, Korf I, Robb S, Parra G, Ross E, Moore B . MAKER: an easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Res. 2007; 18(1):188-96. PMC: 2134774. DOI: 10.1101/gr.6743907. View

Xu X, Nagarajan H, Lewis N, Pan S, Cai Z, Liu X . The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat Biotechnol. 2011; 29(8):735-41. PMC: 3164356. DOI: 10.1038/nbt.1932. View

Fu L, Niu B, Zhu Z, Wu S, Li W . CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012; 28(23):3150-2. PMC: 3516142. DOI: 10.1093/bioinformatics/bts565. View

Li W, Godzik A . Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006; 22(13):1658-9. DOI: 10.1093/bioinformatics/btl158. View

Cohen S, Segal D . Extrachromosomal circular DNA in eukaryotes: possible involvement in the plasticity of tandem repeats. Cytogenet Genome Res. 2009; 124(3-4):327-38. DOI: 10.1159/000218136. View

10.

Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z . clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation (Camb). 2021; 2(3):100141. PMC: 8454663. DOI: 10.1016/j.xinn.2021.100141. View

11.

Stelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M, Fishilevich S . The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses. Curr Protoc Bioinformatics. 2016; 54:1.30.1-1.30.33. DOI: 10.1002/cpbi.5. View

12.

Kaufman R, Sharp P, LATT S . Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences. Mol Cell Biol. 1983; 3(4):699-711. PMC: 368586. DOI: 10.1128/mcb.3.4.699-711.1983. View

13.

Fan Y, Mao R, Lv H, Xu J, Yan L, Liu Y . Frequency of double minute chromosomes and combined cytogenetic abnormalities and their characteristics. J Appl Genet. 2010; 52(1):53-9. DOI: 10.1007/s13353-010-0007-z. View

14.

Yan Y, Guo G, Huang J, Gao M, Zhu Q, Zeng S . Current understanding of extrachromosomal circular DNA in cancer pathogenesis and therapeutic resistance. J Hematol Oncol. 2020; 13(1):124. PMC: 7491193. DOI: 10.1186/s13045-020-00960-9. View

15.

Kazazian Jr H, Moran J . Mobile DNA in Health and Disease. N Engl J Med. 2017; 377(4):361-370. PMC: 5980640. DOI: 10.1056/NEJMra1510092. View

16.

Yu G, Wang L, Han Y, He Q . clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012; 16(5):284-7. PMC: 3339379. DOI: 10.1089/omi.2011.0118. View

17.

Khayami R, Hashemi S, Kerachian M . Role of aldo-keto reductase family 1 member B1 (AKR1B1) in the cancer process and its therapeutic potential. J Cell Mol Med. 2020; 24(16):8890-8902. PMC: 7417692. DOI: 10.1111/jcmm.15581. View

18.

Cao X, Wang S, Ge L, Zhang W, Huang J, Sun W . Extrachromosomal Circular DNA: Category, Biogenesis, Recognition, and Functions. Front Vet Sci. 2021; 8:693641. PMC: 8458813. DOI: 10.3389/fvets.2021.693641. View

19.

Bolger A, Lohse M, Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15):2114-20. PMC: 4103590. DOI: 10.1093/bioinformatics/btu170. View

20.

Moritz B, Becker P, Gopfert U . CMV promoter mutants with a reduced propensity to productivity loss in CHO cells. Sci Rep. 2015; 5:16952. PMC: 4652263. DOI: 10.1038/srep16952. View