CHO cells are most commonly used for the synthesis of recombinant proteins in biopharmaceutical production. When stable producer cell lines are obtained, the locus of transgene integration into the genome has a great influence on the level of its expression. Therefore, the identification of genomic loci ensuring a high level of protein production is very important. Here, we used the TRIP assay to study the influence of the local chromatin environment on the activity of transgenes in CHO cells. For this purpose, reporter constructs encoding eGFP under the control of four promoters were stably integrated into the genome of CHO cells using the piggyBac transposon. Each individual transgene contained a unique tag, a DNA barcode, and the resulting polyclonal cell population was cultured for almost a month without any selection. Next, using the high-throughput sequencing, genomic localizations of barcodes, as well as their abundances in the population and transcriptional activities were identified. In total, ~640 transgenes more or less evenly distributed across all chromosomes of CHO cells were characterized. More than half of the transgenes were completely silent. The most active transgenes were identified to be inserted in gene promoters and 5' UTRs. Transgenes carrying Chinese hamster full-length promoter of the EF-1α gene showed the highest activity. Transgenes with a truncated version of the same promoter and with the mouse PGK gene promoter were on average 10 and 19 times less active, respectively. In total, combinations of genomic loci of CHO cells and transgene promoters that together provide different levels of transcriptional activity of the model reporter construct were described.
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http://dx.doi.org/10.18699/VJGB-23-105 | DOI Listing |
Chembiochem
January 2025
Iryo Sosei University: Iryo Sosei Daigaku, Life Science and Engineering, JAPAN.
Vitamin D receptor (VDR) plays a critical role in regulating multiple biological processes, including bone metabolism and cell differentiation, by mediating transcriptional activation in response to ligand binding. We have constructed an environmentally fluorescent probe 2 for VDR to facilitate real-time observation of its ligand-dependent conformational changes in living cells. This probe 2 was synthesized by introducing a dansyl fluorophore via an ethynyl group at the C11 position of 1α,25(OH)2D3.
View Article and Find Full Text PDFJ Ethnopharmacol
January 2025
Third-grade Pharmacological Laboratory on Traditional Chinese Medicine, State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China; College of Basic Medical Science, China Three Gorges University, Yichang 443002, China. Electronic address:
Ethnopharmacological Relevance: Panax japonicus (T. Nees) C.A.
View Article and Find Full Text PDFInt J Biol Macromol
January 2025
School of Food Science and Technology, Kyungpook National University, Daegu 702-701, Republic of Korea; Research Institute of Tailored Food Technology, Kyungpook National University, Daegu 41566, Republic of Korea. Electronic address:
Zophobas morio protein (ZMP) is a promising protein resource with notable biological properties, and its hydrolysis could unlock enhanced bioactivities. This study investigated ultrasound-assisted enzymatic hydrolysis (UAEH) of ZMP using different enzymes (Alcalase, Neutrase, and Protamex) to determine its effect on the degree of hydrolysis (DH) compared to enzymatic hydrolysis (EH). UAEH showed greater hydrolysis efficiency than EH, with Alcalase exhibiting the highest DH.
View Article and Find Full Text PDFSci Adv
January 2025
Department of Developmental and Regenerative Neurobiology, Institute of Brain Science, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan.
Birth is one of the most important life events for animals. However, its significance in the developmental process is not fully understood. Here, we found that birth-induced alteration of glutamine metabolism in radial glia (RG), the embryonic neural stem cells (NSCs), is required for the acquisition of quiescence and long-term maintenance of postnatal NSCs.
View Article and Find Full Text PDFAppl Microbiol Biotechnol
January 2025
School of Interdisciplinary Research, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India.
Monoclonal antibodies are extensively used as biotherapeutics for treatment of a variety of diseases. Glycosylation of therapeutic antibodies is considered a critical quality attribute as it influences the effector function, circulatory half-life, immunogenicity, and eventually efficacy and patient safety. During upstream process development, media components play a significant role in determining the glycosylation profile.
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