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.
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The selection of clonally derived Chinese hamster ovary (CHO) cell lines with the highest production rate of recombinant glycoproteins remains a big challenge during early stages of cell line development. Different strategies using either product titer or product titer normalized to cell number are being used to assess suspension-adapted clones when grown statically in microtiter plates. However, no reported study so far has performed a direct head-to-head comparison of these two early reporters for predicting clone performance. Therefore, a screening platform for high-throughput analysis of titer and confluence of etanercept-producing clones is developed. Then an unbiased comparison of clone ranking based on either titer or titer normalized to confluence (TTC) is performed. Using two different suspension cultivation vessels, the authors demonstrate that titer- or TTC-based ranking gives rise to the selection of clones with similar volumetric productivity in batch cultures. Therefore, using both titer- and TTC-based ranking is proposed, allowing for selection of distinct clones with both high integral of viable cell density (IVCD) and high specific productivity features, respectively. This contributes to selection of a versatile panel of clones that can be further characterized and from which the final producer clone can be selected that best fits the production requirements.
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