Stability and transcriptomic study of C12orf35 site-specific integrated CHO cell line
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ABSTRACT:
PROVIDER: PRJNA839086 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19256300_1.fastq.gz | Fastqsanger.gz | |||
| SRR19256300_2.fastq.gz | Fastqsanger.gz | |||
| SRR19256301_1.fastq.gz | Fastqsanger.gz | |||
| SRR19256301_2.fastq.gz | Fastqsanger.gz | |||
| SRR19256302_1.fastq.gz | Fastqsanger.gz |
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Project description:Background: Chinese hamster ovary (CHO) cells are the most widely used mammalian host for recombinant protein production. The primary production method is to construct stable, high-yielding cell lines to provide high-quality, low-cost products. The key to optimizing recombinant protein production is to modify engineered cell lines to obtain better growth, expression, or product quality characteristics. Site-specific integration technology can build monoclonal cell lines with ideal stability, yield, and epigenetics by integrating transgenes at stable transcriptional hotspots, becoming a potential tool for constructing genetically engineered cell lines. The previous study screened the C12orf35 site of CHO cells and built an engineered cell line expressing a mAb by CRISPR/Cas9. While the mechanism of site-specific integration of transgenes to achieve high-efficiency expression also needs to be further studied. In this study, we used the extracellular domains of CLL1 and CD33 as model proteins to construct engineered cell lines integrating at C12orf35. Then we studied the growth, expression, and passage stability of these cell lines and conducted transcriptomic studies. Method: In this study, the transgenes CLL1 and CD33 were site-specifically integrated into the C12orf35 site of the CHO-S cell by CRISPR/Cas9 technology. To obtain stable and high-yielding cell lines, we screened and identified monoclonal cell lines by 5’/3’ junction PCR, out-out PCR, and Western blot. Then we compared the growth characteristics of CLL1 and CD33 monoclonal cell lines with wild-type CHO-S cell lines to investigate the growth characteristics. We studied the protein yield and affinity of CLL1 and CD33 cell lines through protein purification, BCA quantification, and ELISA. In addition, we assessed the stability of monoclonal cell lines in long-term culture by studying the growth characteristics, protein yield, and protein quality of early (5th-10th passage) and late (about 20th-25th passage) cells. We performed transcriptomic studies on cell lines with different transgenes, including CLL1, CD33, and anti-PD-1 mAb, inserted at C12orf35. We analyzed the differential genes, GO enrichment, and KEGG enrichment of cell lines hoping to explore the reasons for the phenotypic changes of cell lines from the transcriptional level, as well as the high expression mechanism of C12orf35 site-specific integration cell lines. Results:Using the site-specific integration technology mediated by CRISPR/Cas9, we successfully built CLL1 and CD33 monoclonal cell lines integrated at the C12orf35 site. We evaluated the cell growth characteristics and protein expression properties. The results showed that the insertion of different transgenes at the C12orf35 site had no specific effect on the growth state of the cells. The proteins yields were about 120 mg/L and 160 mg/L, respectively. And the EC50 values of CLL1 and CD33 with the corresponding antibodies reached about 0.36 μg/mL and 0.1 μg/mL, respectively. In addition, the above indicators had similar properties between the 5th and 20th generations, indicating that the specific integration at C12orf35 can achieve both high expression of the target protein and production stability. Transcriptomic analysis showed that the integration of transgenes at the C12orf35 site of CHO cells mainly induced genes in the GO term of “Regulation of transcription, DNA-templated” differentially expressed. Further analysis screened that ATF4 and multiple ZFPs family members were significantly up-regulated. ATF4 and ZFPs are related to gene transcription and protein synthesis, which may be the mechanism of the stable and high expression of the cell lines. Besides, differential gene analysis showed that Car4 and CXCL12 were significantly up- and down-regulated in CD33 cells, respectively. And KEGG enrichment showed that Wnt, PI3K/AKT, and JAK/STAT pathways related to cell proliferation and apoptosis were significantly enriched in CD33 cells. These genes and pathways may be related to the growth state of CD33. In summary, this paper studied the growth characteristics, protein expression characteristics, and stability of the C12orf35 site-specific integration of recombinant CHO cell lines. Combined with transcriptomic analysis, this paper explored the stable and high expression mechanism of these cell lines, hoping to provide a theoretical reference for the subsequent construction and optimization of high-expression stable cell lines.
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Project description:Gene expression is a key determinant of phenotypes that made Chinese Hamster Ovary (CHO) cells, with their human-like glycosylation profile and high protein titers, one of the most widely used cells for the production of therapeutic proteins and biopharmaceuticals. Engineering CHO gene expression thus holds a key to improve drug quality and cost effective production. However, the success of engineering gene expression or ectopic activation of silent genes to optimize desired pathways requires accurate annotation of the underlying regulatory elements and the transcription start site (TSS). Unfortunately, to date, most TSSs of CHO-expressed genes and the ~50% of hamster genes that are silent in CHO were computationally predicted and are frequently inaccurate. To oust this hurdle, we report revised TSSs annotations for 15,308 Chinese Hamster genes and 4,145 non-coding RNAs based on experimental data from CHO K1 cells and 10 hamster tissues. In the example of the glycosyltransferase gene Mgat3, we further demonstrate how accurate annotations readily facilitate activating silent genes by CRISPRa. Together, we envision that our annotation and data from the Chinese Hamster will provide a rich resource for the CHO community, improve genome engineering efforts and additionally aid comparative and evolutionary studies.
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Project description:CHO cells have primarily been studied in bulk, assuming that these bulk samples are identical because of genetic clonality across the sample. In this study, we performed single-cell RNA sequencing on two clonal CHO-K1 cell populations with different stability phenotypes over a 90 day culture period.
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Project description:We report a a large deletion in the gene encoding for laminin subunit α2 (Lama2) in CHO pgsA745.
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Project description:Aim is to validate content of human genomic material in CHO cells monosomal for human chromosome 6. Comparative genomic hybridization to define human genomic regions maintained in hamster hybridoma cell line.
2014-06-05 | E-GEOD-56334 | biostudies-arrayexpress
Project description:Transcriptomic data from CHO-S cells were gathered from multiple time points during batch culture to provide information about gene presence/absence for generation of a CHO-S cell line specific model using the GIMME algorithm.
2016-11-23 | GSE77800 | GEO
Project description:CHO cells are major hosts for the industrial production of therapeutic proteins and their production stability is of considerable economic significance. It is widely known that CHO cells can rapidly acquire genetic alterations, which affects their genetic homogeneity over time. However, the role of non-genetic mechanisms, including epigenetic mechanisms such as DNA methylation, remains poorly understood. We have now used whole-genome bisulfite sequencing to establish single-base methylation maps of eight independent CHO cell lines. Our results identify CpG islands and low-methylated regions as conserved elements with dynamic DNA methylation. Interestingly, methylation patterns were found to cluster clearly along the three main branches of CHO evolution, with no directional changes over short culture periods. Furthermore, multi-ome single-cell sequencing of 9,833 nuclei from three independent cultures revealed dynamic subpopulation structures characterized by robust expression differences in pathways related to protein production. Our findings thus provide novel insights into the epigenetic heterogeneity of CHO cells and support the development of epigenetic biomarkers that trace the emergence of subpopulations in CHO cultures.
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Project description:CHO cells are major hosts for the industrial production of therapeutic proteins and their production stability is of considerable economic significance. It is widely known that CHO cells can rapidly acquire genetic alterations, which affects their genetic homogeneity over time. However, the role of non-genetic mechanisms, including epigenetic mechanisms such as DNA methylation, remains poorly understood. We have now used whole-genome bisulfite sequencing to establish single-base methylation maps of eight independent CHO cell lines. Our results identify CpG islands and low-methylated regions as conserved elements with dynamic DNA methylation. Interestingly, methylation patterns were found to cluster clearly along the three main branches of CHO evolution, with no directional changes over short culture periods. Furthermore, multi-ome single-cell sequencing of 9,833 nuclei from three independent cultures revealed dynamic subpopulation structures characterized by robust expression differences in pathways related to protein production. Our findings thus provide novel insights into the epigenetic heterogeneity of CHO cells and support the development of epigenetic biomarkers that trace the emergence of subpopulations in CHO cultures.
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