10 research outputs found
Transcriptomic signatures classifying CHO Quasispecies
Chinese hamster ovary (CHO) cell lines have the capacity to correctly fold, assemble and modify proteins post-translationally, and consequently is commonly used expression systems for recombinant therapeutic proteins. In recent years, a thorough understanding of process parameters of individual CHO cell lines have been achieved, but comprehending the genomic or pathway-specific distinction of various CHO cell lines at transcriptome level still remains a challenge. To address this challenge i.e. to gain cell line specific understanding of modulation in the pathways and gene sets, an RNA-seq study of CHOS, CHOK1 and DG44 cell lines grown in batch culture has been performed using an in-house developed pipeline. An R-based application was developed specifically for this CHO dataset to further calculate expression values across different cell lines. Furthermore, two main conditions have been defined to perform differential expression (DE) analysis to study regulatory activity across the cell lines. First, we performed a DE analysis of exponential and stationary phase of different cell lines where gene sets having significant DE (p\u3c0.05) was identified. And the second one is DE comparison for both phases disjointedly and collectively over cell lines was conducted and genes with p\u3c0.05 was identified. Among the identified up- and down-regulated genes, unique and common genes across phases and cell lines were identified. Additionally, specific pathways have been found to be regulated similarly across various cell lines and some to be transversely regulated over phases. We have thereby mapped the cell line-specific genetic regulation. This can be implemented in picking desired characters, across various CHO cell lines and in determining the structure of super CHO cell lines having the capability to combat most of the deficiencies exiting till today
Transcriptomic signatures classifying CHO quasispecies
Chinese hamster ovary (CHO) cell lines have the capacity to correctly fold, assemble and modify proteins post-translationally, and consequently is commonly used expression systems for recombinant therapeutic proteins. In recent years, a thorough understanding of process parameters of individual CHO cell lines have been achieved, but comprehending the genomic or pathway-specific distinction of various CHO cell lines at transcriptome level still remains a challenge. To address this challenge i.e. to gain cell line specific understanding of modulation in the pathways and gene sets, an RNA-seq study of CHOS, CHOK1 and DG44 cell lines grown in batch culture was performed using an in-house developed pipeline. An R-based CHO gene expression visualization application was developed specifically for CHO dataset to further visualize expression values across different cell lines. Further, distinction between various CHO cell lines were identified by performing differential expression analysis on some selected pathways related to metabolic and cellular processes. Consequently, most efficient cell lines were picked on the basis of process and pathway specific gene networks. Furthermore, two main conditions i.e. p-value \u3c 0.05 and log fold change of 1 was applied to perform differential expression (DE) analysis to study gene network across the cell lines. Among the identified up- and down-regulated genes, unique and common genes across cell lines were identified. Additionally, specific pathways were found to be similarly regulated and some to be transversely regulated across various cell lines. We have thereby mapped the cell line-specific genetic regulation. This can be implemented in picking desired characters, across various CHO cell lines and in determining the structure of super CHO cell lines having the capability to combat most of the deficiencies exiting till today
An Online Compendium of CHO RNA-Seq Data Allows Identification of CHO Cell Line-specific Transcriptomic Signatures
Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients
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Modular 5′-UTR hexamers for context-independent tuning of protein expression in eukaryotes
Functional characterization of regulatory DNA elements in broad genetic contexts is a prerequisite for forward engineering of biological systems. Translation initiation site (TIS) sequences are attractive to use for regulating gene activity and metabolic pathway fluxes because the genetic changes are minimal. However, limited knowledge is available on tuning gene outputs by varying TISs in different genetic and environmental contexts. Here, we created TIS hexamer libraries in baker's yeast Saccharomyces cerevisiae directly 5' end of a reporter gene in various promoter contexts and measured gene activity distributions for each library. Next, selected TIS sequences, resulted in almost 10-fold changes in reporter outputs, were experimentally characterized in various environmental and genetic contexts in both yeast and mammalian cells. From our analyses, we observed strong linear correlations (R2 = 0.75-0.98) between all pairwise combinations of TIS order and gene activity. Finally, our analysis enabled the identification of a TIS with almost 50% stronger output than a commonly used TIS for protein expression in mammalian cells, and selected TISs were also used to tune gene activities in yeast at a metabolic branch point in order to prototype fitness and carotenoid production landscapes. Taken together, the characterized TISs support reliable context-independent forward engineering of translation initiation in eukaryotes
A Versatile System for USER Cloning-Based Assembly of Expression Vectors for Mammalian Cell Engineering
A new versatile mammalian vector system for protein production, cell biology analyses, and cell factory engineering was developed. The vector system applies the ligation-free uracil-excision based technique--USER cloning--to rapidly construct mammalian expression vectors of multiple DNA fragments and with maximum flexibility, both for choice of vector backbone and cargo. The vector system includes a set of basic vectors and a toolbox containing a multitude of DNA building blocks including promoters, terminators, selectable marker- and reporter genes, and sequences encoding an internal ribosome entry site, cellular localization signals and epitope- and purification tags. Building blocks in the toolbox can be easily combined as they contain defined and tested Flexible Assembly Sequence Tags, FASTs. USER cloning with FASTs allows rapid swaps of gene, promoter or selection marker in existing plasmids and simple construction of vectors encoding proteins, which are fused to fluorescence-, purification-, localization-, or epitope tags. The mammalian expression vector assembly platform currently allows for the assembly of up to seven fragments in a single cloning step with correct directionality and with a cloning efficiency above 90%. The functionality of basic vectors for FAST assembly was tested and validated by transient expression of fluorescent model proteins in CHO, U-2-OS and HEK293 cell lines. In this test, we included many of the most common vector elements for heterologous gene expression in mammalian cells, in addition the system is fully extendable by other users. The vector system is designed to facilitate high-throughput genome-scale studies of mammalian cells, such as the newly sequenced CHO cell lines, through the ability to rapidly generate high-fidelity assembly of customizable gene expression vectors
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Modular 5'-UTR hexamers for context-independent tuning of protein expression in eukaryotes.
Functional characterization of regulatory DNA elements in broad genetic contexts is a prerequisite for forward engineering of biological systems. Translation initiation site (TIS) sequences are attractive to use for regulating gene activity and metabolic pathway fluxes because the genetic changes are minimal. However, limited knowledge is available on tuning gene outputs by varying TISs in different genetic and environmental contexts. Here, we created TIS hexamer libraries in baker's yeast Saccharomyces cerevisiae directly 5' end of a reporter gene in various promoter contexts and measured gene activity distributions for each library. Next, selected TIS sequences, resulted in almost 10-fold changes in reporter outputs, were experimentally characterized in various environmental and genetic contexts in both yeast and mammalian cells. From our analyses, we observed strong linear correlations (R2 = 0.75-0.98) between all pairwise combinations of TIS order and gene activity. Finally, our analysis enabled the identification of a TIS with almost 50% stronger output than a commonly used TIS for protein expression in mammalian cells, and selected TISs were also used to tune gene activities in yeast at a metabolic branch point in order to prototype fitness and carotenoid production landscapes. Taken together, the characterized TISs support reliable context-independent forward engineering of translation initiation in eukaryotes
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A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism
Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways in CHO and associated them with >1,700 genes in the Cricetulus griseus genome. The genome-scale metabolic model based on this reconstruction, iCHO1766, and cell-line-specific models for CHO-K1, CHO-S, and CHO-DG44 cells provide the biochemical basis of growth and recombinant protein production. The models accurately predict growth phenotypes and known auxotrophies in CHO cells. With the models, we quantify the protein synthesis capacity of CHO cells and demonstrate that common bioprocess treatments, such as histone deacetylase inhibitors, inefficiently increase product yield. However, our simulations show that the metabolic resources in CHO are more than three times more efficiently utilized for growth or recombinant protein synthesis following targeted efforts to engineer the CHO secretory pathway. This model will further accelerate CHO cell engineering and help optimize bioprocesses