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.

Project description:Background: Cell line specific, genome-scale metabolic models enable rigorous and systematic in silico investigation of cellular metabolism. Such models have recently become available for Chinese hamster ovary (CHO) too. However, a key ingredient, namely an experimentally validated biomass function that summarizes the cellular composition, was so far missing in all genome-scale metabolic models of CHO cell lines. Here, we close this gap by providing extensive experimental data on the biomass composition of 13 parental and producer CHO cell lines under various conditions.Results: We report protein, lipid, DNA, RNA and carbohydrate content, cell dry mass, and protein and lipid composition. Furthermore, we present meticulous data on exchange rates between cells and environment and provide detailed experimental protocols on how to determine all of the above. The biomass composition is converted into cell line and condition specific biomass functions for usage in cell line specific, genome-scale metabolic models of CHO. Finally, flux-balance analysis (FBA) is used to demonstrate consistency between in silico predictions and experimental analysis.Conclusions: Our study reveals a strong variability of the total protein content and cell dry mass across cell lines. Yet, the relative amino acid composition is independent of the cell lines and conditions and thus needs not be explicitly measured for each new cell line. In contrast, the lipid composition is strongly influenced by the growth media and needs to be determined for each case. These cell line specific variations in the biomass composition have a small impact on growth rate predictions with FBA, as inaccuracies in the predictions are typically dominated by inaccuracies in the exchange rate spectra. Cell specific biomass variations become only important if the experimental errors in the exchange rate spectra drop below twenty percent. Links:All data and code for data processing are available at <a href='http://dx.doi.org/10.17632/g3wz7spyc9.1' rel='noopener noreferrer' target='_blank'>Data Mendeley</a>.Cell line specific metabolic models were deposited in <a href='https://www.ebi.ac.uk/biomodels/(Chelliah et al, 2014)' rel='noopener noreferrer' target='_blank'>BioModels</a>, identifiers are listed in the study sample table.

Project description:Exploiting the full potential of insertional mutagenesis screens with retroviruses and transposons requires methods for distinguishing clonal from subclonal insertion events within heterogeneous tumor cell populations. Current protocols, based on ligation mediated PCR, depend on endonuclease based fragmentation of genomic DNA, resulting in strong biases in amplification and sequencing due to a fixed product sizes of the amplicon. We have developed a method called shear-splink, which enables the semi-quantitative high-throughput sequence analysis of insertional mutations, enabling us to count the number of cells harboring a given integration, within a heterogeneous sample. The shear-splink method enriches for (sub)clonal integrations, thereby reducing the contribution of irrelevant passenger mutations normally hampering a reliable identification of common integration sites. Additionally, this improvement allows us to identify genetic interactions between affected genes, co-occurring mutations and to study acquired resistance mechanisms both in vivo and in vitro. Sequencing of retrovrial integration sites by LM-PCR. The associated manuscript describes a new method to quantitatively determine retrovrial integration sites using an improved ligation-mediated PCR approach and subsequent 454 pyrosequencing. [GSM562151 to GSM562159]: Sequence data from different mixtures of 2 different cell lines (called AE6 and BB12) which are processed without a restriction enzyme. These cell lines are derived from an MMTV induced mammary tumor, for which we amplify the MMTV integration sites using a ligation-mediated PCR setup. We mixed these 2 cell lines, both with a different integration spectrum, to determine whether our amplification and sequencing protocol is quantitative, meaning that the coverage per integration site is decreasing upon a further dilution of the sample. [GSM641935 to GSM641950]: Unique Sleeping beauty induced lymphoma specimens (spleen) obtained from a cohort of 16 wild-type mice with the 129P2/C57BL/6J mixed background. [GSM776576 to GSM776956]: The 379 submitted specimens are originating from 127 unique leukemia/lymphoma samples, processed using 3 different techniques in order to identify Sleeping Beauty integration sites. We compared restriction enzyme based LM-PCR (RE-splink) with shearing based LM-PCR (shear-splink) on 127 unique Sleeping Beauty (SB) induced leukemia's/lymphomas. All sequence data generated by the 454 sequencing platform are submitted to GEO, including the final output of our sequence analysis pipeline (in bed format; see Supplementary files linked below). Previous submissions contained similar sequence information (integration sites of viruses or transposons driving tumorigenesis) and are all part of the same manuscript.

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