Minimizing Clonal Variation during Mammalian Cell Line Engineering for Improved Systems Biology Data Generation

Abstract

Mammalian cells are widely used to express genes for basic biology studies and biopharmaceuticals. Current methods for generation of engineered cell lines introduce high genomic and phenotypic diversity, which hamper studies of gene functions and discovery of novel cellular mechanisms. Here, we minimized clonal variation by integrating a landing pad for recombinase-mediated cassette exchange site-specifically into the genome of CHO cells using CRISPR and generated subclones expressing four different recombinant proteins. The subclones showed low clonal variation with high consistency in growth, transgene transcript levels and global transcriptional response to recombinant protein expression, enabling improved studies of the impact of transgenes on the host transcriptome. Little variation over time in subclone phenotypes and transcriptomes was observed when controlling environmental culture conditions. The platform enables robust comparative studies of genome engineered CHO cell lines and can be applied to other mammalian cells for diverse biological, biomedical and biotechnological applications.

Systems Biology and Multi-Omics Integration: Viewpoints from the Metabolomics Research Community

“…Systems biology is an interdisciplinary research field that requires the combined contribution of chemists, biologists, mathematicians, physicists, and engineers to untangle the biology of complex living systems by integrating multiple types of quantitative molecular measurements with well-designed mathematical models [1,2]. The premise and promise of systems biology has provided a powerful motivation for scientists to combine the data generated from multiple omics approaches (e.g., genomics, transcriptomics, proteomics, and metabolomics) to create a more holistic understanding of cells, organisms, and communities, relating to their growth, adaptation, development, and progression to disease [3,4,5,6,7,8,9,10,11,12].…”

Cell function profiling to assess clone stability

“…Here the ChemStress® time series analysis was interpreted as a novel clone stability metric, but the same approach could be applied whenever cells must be monitored over time, such as continuous bioprocessing, for example. Much has been made of clone stability improvements due to targeted transgene integration, as demonstrated by the transcriptomic studies of Grav et al (Grav et al, 2018), but Chemstress® potentially is an easier way to demonstrate the stability of deep cell function. Broader still, ChemStress® cell function profiling technology could lead to a deeper understanding of how different recombinant products (e.g., easy‐ vs. difficult‐to‐express proteins, product classes), expression engineering strategies, and media components impact upon the cell and cell stability.…”

Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production

“…Therefore, the differences in q P and VCD might be attributed to genomic differences between clones. The same group later addressed this issue with the establishment of an isogenic cell line (Grav et al, 2018).…”

“…However, there is a chance of off‐target effects and of aberrant recombination. The best approach moving forward would be to establish an isogenic cell line using recombinase‐mediated cassette exchange into a stable genomic location (Grav et al, 2018).…”