Identification of transgene integration sites, their structure and epigenetic status with Cas9-targeted nanopore sequencing in CHO cells

Please click Additional Files below to see the full abstract

Profiling lnc-RNA in CHO cells using NGS technologies

Please click Additional Files below to see the full abstract

Profiling the landscape of long non- coding RNAs in the Chinese hamster ovary cell transcriptome

Chinese hamster ovary (CHO) cells are the most commonly used cell line for the production of recombinant proteins in the pharmaceutical industry. Next generation sequencing (NGS) experiments have produced Cricetulus griseus and CHO genomic and transcriptomic references that have expanded the capability to study molecular mechanisms of CHO cells and enabled cell line engineering strategies to enhance industrially relevant phenotypes. The quality of annotation of protein coding genes is comparable to model organisms such as humans and mice. The quality of annotation of long non-coding RNAs (lncRNAs), transcripts longer than 200 nucleotides that do not code for a protein, is not comparable and needs to be expanded in order to fully characterize the C. griseus and CHO genomes. lncRNAs function as regulator molecules that govern the expression of diverse pathways and are ideal targets for future cell line engineering efforts. In this thesis, the genome and transcriptome of CHO cells were profiled in order to discover and characterize lncRNAs. A computational pipeline that can detect and annotate lncRNAs from RNASeq data was constructed and used to relate differential expression to a reduction in culture temperature. A multi-omics experiment was performed which included proteomics and transcriptomics to profile expression and alternative splicing of lncRNAs over the duration of an industrial culture in bioreactors. Next, global chromatin accessibility was profiled in cells that were exposed to a reduction in culture temperature using the Assay for Transposase Accessible Chromatin (ATAC-Seq) in order to identify chromatin-lncRNA regulatory networks. The findings of these studies have expanded the understanding of long non-coding RNAs within CHO cells and provide a resource for future cell line development

Profiling the landscape of long non- coding RNAs in the Chinese hamster ovary cell transcriptome

Chinese hamster ovary (CHO) cells are the most commonly used cell line for the production of recombinant proteins in the pharmaceutical industry. Next generation sequencing (NGS) experiments have produced Cricetulus griseus and CHO genomic and transcriptomic references that have expanded the capability to study molecular mechanisms of CHO cells and enabled cell line engineering strategies to enhance industrially relevant phenotypes. The quality of annotation of protein coding genes is comparable to model organisms such as humans and mice. The quality of annotation of long non-coding RNAs (lncRNAs), transcripts longer than 200 nucleotides that do not code for a protein, is not comparable and needs to be expanded in order to fully characterize the C. griseus and CHO genomes. lncRNAs function as regulator molecules that govern the expression of diverse pathways and are ideal targets for future cell line engineering efforts. In this thesis, the genome and transcriptome of CHO cells were profiled in order to discover and characterize lncRNAs. A computational pipeline that can detect and annotate lncRNAs from RNASeq data was constructed and used to relate differential expression to a reduction in culture temperature. A multi-omics experiment was performed which included proteomics and transcriptomics to profile expression and alternative splicing of lncRNAs over the duration of an industrial culture in bioreactors. Next, global chromatin accessibility was profiled in cells that were exposed to a reduction in culture temperature using the Assay for Transposase Accessible Chromatin (ATAC-Seq) in order to identify chromatin-lncRNA regulatory networks. The findings of these studies have expanded the understanding of long non-coding RNAs within CHO cells and provide a resource for future cell line development