Omics approach for generating a high-yield CHO cell line producing monoclonal antibodies

Chinese hamster ovary (CHO) cells are extensively used for the industrial manufacture of therapeutic antibodies. Generating high producing cell lines for secretory protein production requires knowing the bottleneck in the cellular machinery for protein expression. Integration site of gene of interest (GOI) is one of the important factors that influence the protein productivity. Even though screening of cells randomly integrated GOI can select high producing cells, the selected cell might not stable due to the chromosome instability. Here, we would like to look for host integration sites where GOI is high yield and stable by screening a single copy integration system. We developed several methods to identify integration sites including PCR based, whole genome sequencing based, and a platform to integrate a single copy of GOI into host genome. By determining the integration sites of the high producing clones, we can elucidate the major high yield sites for target gene expression. We have also employed the genome-editing tool, TALEN and CRISPR/cas9 to specifically integrate the vector with an antibody gene into two integration sites of CHO genome. Our data showed, IS1 and IS2 integration sites can be actively edited and specifically integrated an antibody expression vector of 15kb by either TALEN or CRISPR/Cas9. We successfully established site specifically integrated cell pools and expanded the FACS-sorted single cell into a cell line. Each single cell derived cell lines was confirmed by junction-PCR and sequence analysis. Furthermore, these single cells derived CHO cell lines are shown to express antibody gene with high titer. With the combination of omics knowledge and toolbox, including CHO genomics, transcriptomics and CHO specific microarray, GOI can be stably and highly produced

High-yield antibody production using targeted integration and engineering CHO host

To identify the high expression sites in the CHO cells, we employed NGS to analyze the integration sites of a high producing cell line (titer \u3e 3g/L). The pair-end reads with one read mapped to the vector and the other read mapped to the CHO reference genome are extracted to identify the integration sites. To test the expression activity of the integration sites, we employed CRISPR/Cas9 to specifically integrate the antibody gene into CHO genome for expression. Our data showed 4 integration sites are in the high producing cell line. Among the 4 integration site, one integration site was tested by CRISPR/Cas9 for target integration of antibody gene for expression. The target integrated cell pool present higher expression level (130 mg/L/copy) and less copy number when compared other integration sites. Through single-copy integration method, we can also achieve 60-150 mg/L/copy in a batch culture. About 80% of the single-copy cell clones were stable at generation 60. We have also applied the CHO-specific microarray transcriptomics technology to identify genes that contribute to high productivity. Transfection of our proprietary dual promoter vector J 1.0 resulting in 1.65 to 2.4 fold increase in the expression in engineered CHO DXB11 host. Through fed-batch process development, 3 – 5 g/L mAb productivity can be achieved through targeted integration and engineered CHO host

Development of high-producing CHO cell lines through target-designed strategy

Productivity and stability are critical for the protein drug producing cell lines for manufacturing. Given that the integration sites of gene of interest (GOI) could contribute remarkable effect on the productivity and stability of GOI expression, we intended to develop a targeting-designed approach to generate the high-producing cell lines in a time-saving and less labor-intensive method through targeting the active and stable regions. To identify the active and stable regions located in CHO genome, two approaches were applied in our experiments. Firstly, the integration sites of GOI in cell clones developed by random integration were identified by whole genome sequencing. Secondly, we developed transposon-mediated low copy integration to discover novel active region located in CHO genome. It is interesting that the productivity per integrated GOI in cell clones developed by transposon system was more than two times to that in cell clones developed by random integration (random integration: 20-40 mg/L/copy; transposon-mediated integration: 40-140mg/L/copy). In addition, about 80% of cell clones developed by transposon system maintained the stability of antibody titer after culturing for 60 generations. These results implied that the potential active and stable integration region in the cell clones developed by transposon system. The identified integration regions could be applied for target integration. In order to verify the expression activity and stability of the integration sites, we employed CRISPR/Cas9 to specifically integrate the antibody gene into CHO genome for expression. Our data showed the cell pool generated by knock-in of expression vector into the IS1 integration site present higher expression titer than cell pools generated by integration into other sites or random integration. We further cultured the single cell clones derived from this cell pool by Clonepix and limiting dilution. These single cell clones have high expression titer ranging from 254 to 804 mg/L in batch culture of after 6 Days. A single cell clone(376 mg/L in batch culture) can reached 2 g/L in fed-batch culture. The stability analysis showed this clone maintain stable expression of GOI after 60 generation. Here, we demonstrated the generation of stable cell line with high protein expression by CRISPR/Cas9 mediated target integration. This approach will cost less time and labor than traditional method

SARAS-Net: Scale and Relation Aware Siamese Network for Change Detection

Change detection (CD) aims to find the difference between two images at different times and output a change map to represent whether the region has changed or not. To achieve a better result in generating the change map, many State-of-The-Art (SoTA) methods design a deep learning model that has a powerful discriminative ability. However, these methods still get lower performance because they ignore spatial information and scaling changes between objects, giving rise to blurry boundaries. In addition to these, they also neglect the interactive information of two different images. To alleviate these problems, we propose our network, the Scale and Relation-Aware Siamese Network (SARAS-Net) to deal with this issue. In this paper, three modules are proposed that include relation-aware, scale-aware, and cross-transformer to tackle the problem of scene change detection more effectively. To verify our model, we tested three public datasets, including LEVIR-CD, WHU-CD, and DSFIN, and obtained SoTA accuracy. Our code is available at https://github.com/f64051041/SARAS-Net

Soil Erosion Modeling and Comparison Using Slope Units and Grid Cells in Shihmen Reservoir Watershed in Northern Taiwan

Soil erosion is a global problem that will become worse as a result of climate change. While many parts of the world are speculating about the effect of increased rainfall intensity and frequency on soil erosion, Taiwan’s mountainous areas are already facing the power of rainfall erosivity more than six times the global average. To improve the modeling ability of extreme rainfall conditions on highly rugged terrains, we use two analysis units to simulate soil erosion at the Shihmen reservoir watershed in northern Taiwan. The first one is the grid cell method, which divides the study area into 10 m by 10 m grid cells. The second one is the slope unit method, which divides the study area using natural breaks in landform. We compared the modeling results with field measurements of erosion pins. To our surprise, the grid cell method is much more accurate in predicting soil erosion than the slope unit method, although the slope unit method resembles the real terrains much better than the grid cell method. The average erosion pin measurement is 6.5 mm in the Shihmen reservoir watershed, which is equivalent to 90.6 t ha−1 yr−1 of soil erosion

Experimental and Simulated Investigations of Thin Polymer Substrates with an Indium Tin Oxide Coating under Fatigue Bending Loadings

Stress-induced failure is a critical concern that influences the mechanical reliability of an indium tin oxide (ITO) film deposited on a transparently flexible polyethylene terephthalate (PET) substrate. In this study, a cycling bending mechanism was proposed and used to experimentally investigate the influences of compressive and tensile stresses on the mechanical stability of an ITO film deposited on PET substrates. The sheet resistance of the ITO film, optical transmittance of the ITO-coated PET substrates, and failure scheme within the ITO film were measured to evaluate the mechanical stability of the concerned thin films. The results indicated that compressive and tensile stresses generated distinct failure schemes within an ITO film and both led to increased sheet resistance and optical transmittance. In addition, tensile stress increased the sheet resistance of an ITO film more easily than compressive stress did. However, the influences of both compressive and tensile stress on increased optical transmittance were demonstrated to be highly similar. Increasing the thickness of a PET substrate resulted in increased sheet resistance and optical transmittance regardless of the presence of compressive or tensile stress. Moreover, J-Integral, a method based on strain energy, was used to estimate the interfacial adhesion strength of the ITO-PET film through the simulation approach enabled by a finite element analysis

Osthole Antagonizes Microglial Activation in an NRF2-Dependent Manner

Microglia are neuroglia in the brain with an innate immune function and participate in the progress of neurodegenerative diseases. Osthole (OST) is a coumarin derivative extracted from Cnidium monnieri and bears a microglia-antagonizing ability. However, the underlying mechanism of the antagonism is not clear. The lipopolysaccharides-induced microglial BV2 cell line and amyloid-overexpressing fruit fly were used as models to study OST treatment. We found that OST treatment is sufficient to evoke NRF2 cascade under an LPS-induced inflammatory environment, and silencing NRF2 is sufficient to abolish the process. Moreover, we found that OST is sufficient to antagonize microglial activation in both LPS-induced BV2 cells and Aβ-overexpressing fruit flies, and silencing NRF2 abolishes OST’s antagonism. Furthermore, OST treatment rescued survival, climbing, and the learning ability of Aβ-overexpressing fruit flies and relieved oxidative stress. In conclusion, we proved that OST antagonizes microglial activation induced by either LPS or Aβ and that NRF2 is necessary for OST’s antagonism