85 research outputs found
Generation of desirable CHO cell factories with predictive culture performance using CRISPR/Cas9-mediated genome engineering
Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus, genome engineering of CHO cells for improved product quality and yield is of great interest. Here, I will demonstrate our latest advances in improving the efficiency of CRISPR/Cas9-mediated genome engineering to generate attractive knockout and knockin CHO cell lines. Analysis of the dynamics and efficiency of the technology will be demonstrated for genes involved in glycosylation and apoptosis. Combined with multiplexing and fluorescent enrichment, application of CRISPR/Cas9 genome editing facilitated disruption of several genes simultaneously and accelerated analysis of gene combinations. Engineered CHO cell lines with multiple disruptions of genes involved in apoptosis and glycosylation showed prolonged growth and improved glycosylation profiles. Site-specific integration of transgenes mediated by CRISPR/Cas9 and homology directed repair facilitated generation of targeted integrants with improved clonal homogeneity compared to random integrants. Improvements in the efficiency of our targeted integration platform combined with identification of good integration sites has facilitated precise insertion and expression of genes encoding biopharmaceuticals. In the end, characterization of engineered CHO cell lines with desirable properties generated using combinations of gene disruptions and insertions will be presented. The proven efficacy of genome engineering mediated by CRISPR/Cas9 technology has a large potential to accelerate current CHO engineering efforts. Together with high-throughput technologies, computational models and systems biology approaches, genome editing can pave the way for accelerated generation of desirable CHO cell factories with predictive culture performance
Generation of a Chinese Hamster Ovary cell genome-wide deletion library
Nowadays, around 70% of all industrially produced biopharmaceuticals are generated from Chinese Hamster Ovary (CHO) cells showing the high interest for further characterization and optimization of this cell line and its derivates. Despite their importance, the connection between the CHO cell genome sequence and function has not been explored in detail so far. Forward genetic screens are the state-of-the-art approach to investigate the link between genotype and phenotype using the CRISPR system as an efficient tool for this purpose. These screens are usually focusing on the ~ 28,000 protein coding genes, which cover only ~ 3 % of the genome. Our approach aims to correlate larger functional regions of the genome, including coding and non-coding sequences, with process relevant cell behavior, such as growth and productivity. To this end, we designed a deletion library approach that targets larger genomic regions of 100 – 150 kb using paired CRISPR gRNAs. So far, we demonstrated successful and efficient deletions up to 150 kb, resulting in proper loss-of-function mutations. These modifications were analyzed on genome and phenotype level, demonstrating that deletion efficiencies are size independent. Furthermore, to enable the presence of active gRNA pairs in each individual cell, we implemented bicistronic transcription of gRNAs separated by a tRNA sequence that unequivocally links each pair. Additionally, we determined CRISPR Cpf1 – an alternative CRISPR enzyme – activity in CHO with no cross-interaction to the CRISPR/Cas9 system, providing the possibility to use the two systems in parallel, one for targeted insertion of the gRNA pair into the genome for later identification of the deleted region, the other for deletion of the corresponding genomic region itself. Currently we are working on the generation of a first smallscale deletion library targeting lncRNAs in CHO for the implementation of the strategy before going genomewide. This will then open the opportunity both of generating large scale gene knockout libraries and of characterizing non-coding genomic regions, gene clusters or regulatory elements
Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions
USP7 counteracts SCFβTrCP- but not APCCdh1-mediated proteolysis of Claspin
Claspin is an adaptor protein that facilitates the ataxia telangiectasia and Rad3-related (ATR)-mediated phosphorylation and activation of Chk1, a key effector kinase in the DNA damage response. Efficient termination of Chk1 signaling in mitosis and during checkpoint recovery requires SCFβTrCP-dependent destruction of Claspin. Here, we identify the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of Claspin stability. Claspin and USP7 interact in vivo, and USP7 is required to maintain steady-state levels of Claspin. Furthermore, USP7-mediated deubiquitylation markedly prolongs the half-life of Claspin, which in turn increases the magnitude and duration of Chk1 phosphorylation in response to genotoxic stress. Finally, we find that in addition to the M phase–specific, SCFβTrCP-mediated degradation, Claspin is destabilized by the anaphase-promoting complex (APC) and thus remains unstable in G1. Importantly, we demonstrate that USP7 specifically opposes the SCFβTrCP- but not APCCdh1-mediated degradation of Claspin. Thus, Claspin turnover is controlled by multiple ubiquitylation and deubiquitylation activities, which together provide a flexible means to regulate the ATR–Chk1 pathway
A new view of responses to first-time barefoot running.
We examined acute alterations in gait and oxygen cost from shod-to-barefoot running in habitually-shod well-trained runners with no prior experience of running barefoot. Thirteen runners completed six-minute treadmill runs shod and barefoot on separate days at a mean speed of 12.5 km·h-1. Steady-state oxygen cost in the final minute was recorded. Kinematic data were captured from 30-consecutive strides. Mean differences between conditions were estimated with 90% confidence intervals. When barefoot, stride length and ground-contact time decreased while stride rate increased. Leg-and vertical stiffness and ankle-mid-stance dorsi-flexion angle increased when barefoot while horizontal distance between point of contact and the hip decreased. Mean oxygen cost decreased in barefoot compared to shod running (90% CI -11% to -3%) and was related to change in ankle angle and point-of-contact distance, though individual variability was high (-19% to +8%). The results suggest that removal of shoes produces an alteration in running gait and a potentially-practically-beneficial reduction in mean oxygen cost of running in trained-habitually-shod runners new to running barefoot. However, high variability suggests an element of skill in adapting to the novel task and that caution be exercised in assuming the mean response applies to all runners
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