Targeted epigenetic glyco-engineering in CHO cells

Extensive knowledge has been gathered by applying and generating –omics techniques and data towards a holistic understanding of the Chinese Hamster Ovary (CHO) cell’s regulatory network. However, these data are far from universally explanatory. The epigenome, i.e. the genetic signature that controls modulation of gene expression, has not yet been fully explored. To enable direct control of epigenetic regulation of individual genes, we constructed CRISPR-based epigenetic editing tools that induce site-specific DNA methylation or demethylation rather than double strand breaks at specific endogenous promoters. The current design targets the promoter of the silenced α (2,6)-sialyltransferase (ST6GAL1) gene, which is actively transcribed in human and there part of the protein glycosylation machinery. It is present in the CHO genome, but silenced. We aimed to induce its expression in CHO by targeted demethylation of the ST6GAL1 promoter. Flow cytometric analysis (based on glycosylation specific lectin staining) showed upregulation of ST6GAL1 in up to 67% of cells in transfected cell pools. ST6GAL1 expression was also confirmed by RT-qPCR and MS glycan analysis. Stable upregulation of ST6GAL1 was monitored over a period of more than 80 days, showing the applicability in industrial cell development pipelines for long-term changes in cell behavior. The effect could be readily reversed by subsequent targeted re-methylation of the ST6GAL1 promoter by our epigenetic editing tool set. In conclusion, this epigenetic tool does not only allow to build a new layer of cell control that complements existing techniques (e.g. genome engineering), but also enables a more sensitive investigation of gene function by induction and repression of genes without altering the DNA sequence. Finally, other than gene knockout or overexpression studies, the modulation is readily reversible, thus opening up a multitude of possibilities for fast, unbiased and stable testing of gene function and multiplex engineering approaches

Targeted DNA methylation of endogenous promoters and the CMV promoter entails distinct and subsequent histone modification changes in CHO cells

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Epigenetic regulation of gene expression in response to a changing environment in CHO cell batch culture

Chinese Hamster Ovary (CHO) cells have been the workhorse for industrial production of recombinant therapeutic proteins since 1987. Variations in cellular environment and phenotypes that occur throughout the bioprocess can bring about significant changes in productivity and quality of recombinant proteins. This can potentially lead to rejection of the production lot. Hence, there is interest in an in-depth understanding of cell-line behavior and control to achieve more predictable and reliable process performance. Biological systems undergo dynamic changes over time, where individual genes are turned “on”, “off” or “paused” as and when required. So far, there is very little information available for CHO cell lines, that elucidates the effect of dynamic epigenetic regulation on temporal expression of genes in response to altered substrate availability and culture conditions. While DNA methylation levels around TSS induce either expression or silencing of genes, transcriptional regulation is primarily considered to be an interplay of transcription factors and chromatin modifiers. On top of these, there is a rapid increase in indications that connects phase-specific long non-coding RNAs (lncRNAs) in transcriptional and post-transcriptional gene regulation. Unfortunately, the mechanism of interaction of these lncRNAs with coding genes have not been studied extensively. In this study, the gene transcription dynamics throughout a batch culture of CHO cells was examined by analyzing expression profiles and histone modifications in regular 12-24 hour intervals. Chromatin states and differential methylation profiles were used to understand the role of epigenetic modifiers in the regulation of gene expression. A good correlation between expression level and absence of DNA-methylation in the promoter regions was observed. Genes having all essential active chromatin marks - specific for promoter activity, genic enhancer and active transcription, also showed significantly high positive correlation between the changes in expression levels and histone marks. Both transcription and chromatin modifications during different growth phases were found to be highly dynamic. Clusters of genes showing similar trends of expression depict gradual and continuous adaptation to the changing substrate concentrations. Less narrowly spaced temporal analyses would have prevented detection of critical regulators involved in transient changes during the batch culture. Here, we also report a plausible mode of interaction of lncRNAs with the coding genes mediated by RNA-DNA-DNA triplex formations. Based on the identified interactions, we could predict possible gene targets and the target sites for the expressed lncRNAs and show high level of correlation of expression levels between interacting pairs. To the best of our knowledge this is the first and most comprehensive report of genome wide transcriptional regulation by epigenetic modifiers for CHO. Please click Additional Files below to see the full abstract

What\u27s in a Phenotype?

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Development and standardisation of Laghu Sudarshan Churna – An Ayurvedic polyherbal formulation

106-112Laghu Sudarshan Churna, LSC is an Ayurvedic polyherbal formulation employed for different types of jvaras (fevers). The present study was undertaken to prepare its standardised formulation and to standardise the finished product using quality control procedures mentioned in Ayurvedic Pharmacopoeia of India (API). For this, four batches of the finished products were prepared on a laboratory scale and performed the pharmacognostical parameters (macroscopic, microscopic and powder drug analysis); thin layer chromatography; quantitative physicochemical evaluation including loss on drying, total ash, acid-insoluble ash, alcohol & water soluble extractive values, and pH; & measuring the level of aflatoxins, microbial load, heavy metals and pesticide residues of the finished product. This study is the foremost effort to develop the standardised formulation along with the evaluation parameters for LSC. Thus, obtained results would be beneficial and will act as the reference for the standardisation of LSC

A reference genome for the Chinese hamster based on a hybrid assembly strategy

Accurate and complete genome sequences are essential in biotechnology, especially to facilitate genome-based cell engineering efforts for the main production host of biotherapeutic proteins, Chinese hamster ovary (CHO) cells. While genome-enabled CHO cell engineering efforts promise to enhance drug production, the current genome assemblies for Cricetulus griseus, the Chinese hamster, are highly fragmented and replete with gap sequences and misassemblies, consistent with most short-read based assemblies. Here we have completely re-sequenced the Chinese hamster, C. griseus, using Single Molecule Real Time (SMRT) long-read technology and merged this assembly with Illumina-based assemblies. Please download the file below for full content

zflncRNApedia: A Comprehensive Online Resource for Zebrafish Long Non-Coding RNAs

<div><p>Recent transcriptome annotation using deep sequencing approaches have annotated a large number of long non-coding RNAs in zebrafish, a popular model organism for human diseases. These studies characterized lncRNAs in critical developmental stages as well as adult tissues. Each of the studies has uncovered a distinct set of lncRNAs, with minor overlaps. The availability of the raw RNA-Seq datasets in public domain encompassing critical developmental time-points and adult tissues provides us with a unique opportunity to understand the spatiotemporal expression patterns of lncRNAs. In the present report, we created a catalog of lncRNAs in zebrafish, derived largely from the three annotation sets, as well as manual curation of literature to compile a total of 2,267 lncRNA transcripts in zebrafish. The lncRNAs were further classified based on the genomic context and relationship with protein coding gene neighbors into 4 categories. Analysis revealed a total of 86 intronic, 309 promoter associated, 485 overlapping and 1,386 lincRNAs. We created a comprehensive resource which houses the annotation of lncRNAs as well as associated information including expression levels, promoter epigenetic marks, genomic variants and retroviral insertion mutants. The resource also hosts a genome browser where the datasets could be browsed in the genome context. To the best of our knowledge, this is the first comprehensive resource providing a unified catalog of lncRNAs in zebrafish. The resource is freely available at URL: <a href="http://genome.igib.res.in/zflncRNApedia" target="_blank">http://genome.igib.res.in/zflncRNApedia</a></p></div

Matrix reporting sample count of various datasets used across different developmental time-points.

<p>Matrix reporting sample count of various datasets used across different developmental time-points.</p

Workflow detailing data curation and methodologies involved in building the resource.

<p>Workflow detailing data curation and methodologies involved in building the resource.</p

Development and standardisation of Laghu Sudarshan Churna – An Ayurvedic polyherbal formulation

Laghu Sudarshan Churna, LSC is an Ayurvedic polyherbal formulation employed for different types of jvaras (fevers). The present study was undertaken to prepare its standardised formulation and to standardise the finished product using quality control procedures mentioned in Ayurvedic Pharmacopoeia of India (API). For this, four batches of the finished products were prepared on a laboratory scale and performed the pharmacognostical parameters (macroscopic, microscopic and powder drug analysis); thin layer chromatography; quantitative physicochemical evaluation including loss on drying, total ash, acid-insoluble ash, alcohol &amp; water soluble extractive values, and pH; &amp; measuring the level of aflatoxins, microbial load, heavy metals and pesticide residues of the finished product. This study is the foremost effort to develop the standardised formulation along with the evaluation parameters for LSC. Thus, obtained results would be beneficial and will act as the reference for the standardisation of LSC