Synergistic promotion of transient transgene expression in CHO cells by PDI/XBP-1s co-transfection and mild hypothermia.

Transient gene expression system is an important tool for rapid production of recombinant proteins in Chinese hamster ovary (CHO) cells. However, their low productivity is the main hurdle to overcome. An effective approach through which to obtain high protein yield involves targeting transcriptional, post-transcriptional events (PTEs), and culture conditions. Here, we investigated the effects of protein disulfide isomerase (PDI) and spliced X-box binding protein 1 (XBP-1s) co-overexpression combined with mild hypothermia on the transient yields of recombinant proteins in CHO cells. The results showed that the gene of interest (GOI) and the PDI/XBP-1s helper vector at a co-transfection ratio of 10:1 could obviously increase transient expression level of recombinant protein in CHO cells. However, PDI/XBP-1s overexpression had no significance effect on the mRNA levels of the recombinant protein, suggesting that it targeted PTEs. Moreover, the increased production was due to the enhancing of cell specific productivity, not related to cell growth, viability, and cell cycle. In addition, combined PDI/XBP-1s co-overexpression and mild hypothermia could further improve Adalimumab expression, compared to the control/37 °C and PDI/XBP-1s/37 °C, the Adalimumab volume yield of PDI/XBP-1s/33 °C increased by 203% and 142%, respectively. Mild hypothermia resulted in 3.52- and 2.33-fold increase in the relative mRNA levels of PDI and XBP-1s, respectively. In conclusion, the combination of PDI/XBP-1s overexpression and culture temperature optimization can achieve higher transient expression of recombinant protein, which provides a synergetic strategy to improve transient production of recombinant protein in CHO cells.

Research progress on the regulation of oxidative stress by phenolics: the role of gut microbiota and Nrf2 signaling pathway.

In recent years, the increase in high-calorie diets and sedentary lifestyles has made obesity a global public health problem. An unbalanced diet promotes the production of proinflammatory cytokines and causes redox imbalance in the body. Phenolics have potent antioxidant activity and cytoprotective ability. They can scavenge free radicals and reactive oxygen species, and enhance the activity of antioxidant enzymes, thus combating the body's oxidative stress. They can also improve the body's inflammatory response, enhance the enzyme activity of lipid metabolism, and reduce the contents of cholesterol and triglyceride. Most phenolics are biotransformed and absorbed into the blood after the action by gut microbiota; these metabolites then undergo phase I and II metabolism and regulate oxidative stress by scavenging free radicals and increasing expression of antioxidant enzymes. Phenolics induce the expression of genes encoding antioxidant enzymes and phase II detoxification enzymes by stimulating Nrf2 to enter the nucleus and bind to the antioxidant response element after uncoupling from Keap1, thereby promoting the production of antioxidant enzymes and phase II detoxification enzymes. The absorption rate of phenolics in the small intestine is extremely low. Most phenolics reach the colon, where they interact with the microbiota and undergo a series of metabolism. Their metabolites will reach the liver via the portal vein and undergo conjugation reactions. Subsequently, the metabolites reach the whole body to exert biological activity by traveling with the systemic circulation. Phenolics can promote the growth of probiotics, reduce the ratio of Firmicutes/Bacteroidetes (F/B), and improve intestinal microecological imbalance. This paper reviews the nutritional value, bioactivity, and antioxidant mechanism of phenolics in the body, aiming to provide a scientific basis for the development and utilization of natural antioxidants and provide a reference for elucidating the mechanism of action of phenolics for regulating oxidative stress in the body. © 2023 Society of Chemical Industry.

Impairment of Endothelial Function by Cigarette Smoke Is Not Caused by a Specific Smoke Constituent, but by Vagal Input From the Airway.

BACKGROUND: Exposure to tobacco or marijuana smoke, or e-cigarette aerosols, causes vascular endothelial dysfunction in humans and rats. We aimed to determine what constituent, or class of constituents, of smoke is responsible for endothelial functional impairment. METHODS: We investigated several smoke constituents that we hypothesized to mediate this effect by exposing rats and measuring arterial flow-mediated dilation (FMD) pre- and post-exposure. We measured FMD before and after inhalation of sidestream smoke from research cigarettes containing normal and reduced nicotine level with and without menthol, as well as 2 of the main aldehyde gases found in both smoke and e-cigarette aerosol (acrolein and acetaldehyde), and inert carbon nanoparticles. RESULTS: FMD was reduced by all 4 kinds of research cigarettes, with extent of reduction ranging from 20% to 46% depending on the cigarette type. While nicotine was not required for the impairment, higher nicotine levels in smoke were associated with a greater percent reduction of FMD (41.1±4.5% reduction versus 19.2±9.5%; P=0.047). Lower menthol levels were also associated with a greater percent reduction of FMD (18.5±9.8% versus 40.5±4.8%; P=0.048). Inhalation of acrolein or acetaldehyde gases at smoke-relevant concentrations impaired FMD by roughly 50% (P=0.001). However, inhalation of inert carbon nanoparticles at smoke-relevant concentrations with no gas phase also impaired FMD by a comparable amount (P<0.001). Bilateral cervical vagotomy blocked the impairment of FMD by tobacco smoke. CONCLUSIONS: There is no single constituent or class of constituents responsible for acute impairment of endothelial function by smoke; rather, we propose that acute endothelial dysfunction by disparate inhaled products is caused by vagus nerve signaling initiated by airway irritation.

Maternal and neonatal outcomes in women with twin pregnancies based on gestational weight gain: An updated systematic review and meta-analysis.

Objective: This updated systematic review and meta-analysis aimed to assess maternal and fetal outcomes of pregnancies based on the Institute of Medicine (IOM) guidelines of gestational weight gain (GWG). Methods: PubMED, SCOPUS, EMBASE and Web of Science were searched up to 30th July 2022. All studies evaluating maternal and/or neonatal outcomes of twin pregnancies based on the IOM guidelines of gestational weight gain were included. Results: Twenty two studies were included. Mothers with twin pregnancies experiencing inadequate GWG showed higher incidence of gestational diabetes with the risk ratio (RR) 1.22 95% CI (0.95,1.57), p=0.0005, i2= 69% and premature rupture of membrane (PROM) with RR 1.14 95% CI (0.99, 1.30), p=0.07; i2=0% that coincided with higher rates of preterm birth, low birth weight, small for gestational age (SGA) and admission to NICU in neonates. Mothers with excessive GWG had higher risk of developing gestational hypertension with RR 1.59 95% CI (1.22, 2.07), p=0.0006, i2=75% and extremely preterm delivery (<32 weeks). Conclusion: Within the limitations of this review, GWG was found to be a predictable risk factor for adverse maternal and neonatal outcomes of twin pregnancies.

Single-shot multi-parametric mapping based on multiple overlapping-echo detachment (MOLED) imaging.

Multi-parametric quantitative magnetic resonance imaging (mqMRI) allows the characterization of multiple tissue properties non-invasively and has shown great potential to enhance the sensitivity of MRI measurements. However, real-time mqMRI during dynamic physiological processes or general motions remains challenging. To overcome this bottleneck, we propose a novel mqMRI technique based on multiple overlapping-echo detachment (MOLED) imaging, termed MQMOLED, to enable mqMRI in a single shot. In the data acquisition of MQMOLED, multiple MR echo signals with different multi-parametric weightings and phase modulations are generated and acquired in the same k-space. The k-space data is Fourier transformed and fed into a well-trained neural network for the reconstruction of multi-parametric maps. We demonstrated the accuracy and repeatability of MQMOLED in simultaneous mapping apparent proton density (APD) and any two parameters among T2, T2*, and apparent diffusion coefficient (ADC) in 130-170 ms. The abundant information delivered by the multiple overlapping-echo signals in MQMOLED makes the technique potentially robust to system imperfections, such as inhomogeneity of static magnetic field or radiofrequency field. Benefitting from the single-shot feature, MQMOLED exhibits a strong motion tolerance to the continuous movements of subjects. For the first time, it captured the synchronous changes of ADC, T2, and T1-weighted APD in contrast-enhanced perfusion imaging on patients with brain tumors, providing additional information about vascular density to the hemodynamic parametric maps. We expect that MQMOLED would promote the development of mqMRI technology and greatly benefit the applications of mqMRI, including therapeutics and analysis of metabolic/functional processes.

Old wine in new bottles: Kaempferol is a promising agent for treating the trilogy of liver diseases.

As a source of various compounds, natural products have long been important and valuable for drug development. Kaempferol (KP) is the most common flavonol with bioactive activity and has been extracted from many edible plants and traditional Chinese medicines. It has a wide range of pharmacological effects on inflammation, oxidation, and tumour and virus regulation. The liver is an important organ and is involved in metabolism and activity. Because the pathological process of liver diseases is extremely complicated, liver diseases involving ALD, NASH, liver fibrosis, and HCC are often complicated and difficult to treat. Fortunately, there have been many reports that KP has a good pharmacological effect on a series of complex liver diseases. To fully understand the mechanism of KP and provide new ideas for its clinical application in the treatment of liver diseases, this article reviews the pharmacological mechanism and potential value of KP in different studies involving various liver diseases. In the trilogy of liver disease, high concentrations of ROS stimulate peroxidation and activate the inflammatory signal cascade, which involves signalling pathways such as MAPK/JAK-STAT/PERK/Wnt/Hipp, leading to varying degrees of cell degradation and liver damage. The development of liver disease is promoted in an inflammatory environment, which is conducive to the activation of TGF-ß1, leading to increased expression of pro-fibrosis and pro-inflammatory genes. Inflammation and oxidative stress promote the formation of tumour microenvironments, and uncontrolled autophagy of cancer cells further leads to the development of liver cancer. The main pathway in this process is AMPK/PTEN/PI3K-Akt/TOR. KP can not only protect liver parenchymal cells through a variety of antioxidant and anti-apoptotic mechanisms but also reduces the immune inflammatory response in the liver microenvironment, thereby preventing cell apoptosis; it can also inhibit the ER stress response, prevent inflammation and inhibit tumour growth. KP exerts multiple therapeutic effects on liver disease by regulating precise signalling targets and is expected to become an emerging therapeutic opportunity to treat liver disease in the future.

Recombinant antibodies aggregation and overcoming strategies in CHO cells.

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modifications similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies (RTAs) are among the most important and promising RTPs for biomedical applications. A major limitation associated with the use of RTAs is their aggregation, which can be caused by a variety of factors; this results in a reduction of quality. RTA aggregations are especially concerning as they can trigger human immune responses in humans and may be fatal. Therefore, the mechanisms underlying RTA aggregation and measures for avoiding aggregation are interesting topics in RTAs research. In this review, we discuss recent progress in the field of RTAs aggregation, with a focus on factors that cause aggregation during RTA production and the development of strategies for overcoming RTA aggregation. KEY POINTS: • The recombinant antibody aggregation in mammalian cell systems is reviewed. • Intracellular environment and extracellular parameters influence recombinant antibody aggregation. • Reducing the aggregations can improve the quality of recombinant antibodies.

Effects and mechanisms of animal-free hydrolysates on recombination protein yields in CHO cells.

Chinese hamster ovary (CHO) cells are the commonly used cell lines for producing recombinant therapeutic proteins (RTPs) because they possess post-translational modifications similar to human cells. Culture media are necessary for cell growth, and their quality affects the yields and quality of RTPs. Due to safety concerns for the complex purification of RTPs, the development of serum-free media (SFM) is necessary for CHO cells. To meet the need for CHO cells with higher cell density and RTP productivity with consistent product quality in large-scale suspension cultures, the optimization of SFM through adding some enzymatic animal-free hydrolysates (AFHs) is preferred. The AFHs can improve cell culture performance and product yield of RTPs without affecting their quality. Here, the effect and mechanism of various AFHs in improving CHO cell culture performance and protein expression are reviewed. KEY POINTS: • AFHs that improve the recombinant protein yield of CHO cells are reviewed. • AFHs improve recombinant protein yield via influencing cell performance. • The AFHs do not affect the quality of recombinant protein in CHO cells. • AFHs can provide nutrients, block cell cycle, and reduce oxidative stress.

Cognitive impairment caused by hypoxia: from clinical evidences to molecular mechanisms.

Hypoxia is a state of reduced oxygen supply and excessive oxygen consumption. According to the duration of hypoxic period, it can be classified as acute and chronic hypoxia. Both acute and chronic hypoxia could induce abundant neurological deficits. Although there have been significant advances in the pathophysiological injuries, few studies have focused on the cognitive dysfunction. In this review, we focused on the clinical evidences and molecular mechanisms of cognitive impairment under acute and chronic hypoxia. Hypoxia can impair several cognitive domains such as attention, learning and memory, procession speed and executive function, which are similar in acute and chronic hypoxia. The severity of cognitive deficit correlates with the duration and degree of hypoxia. Recovery can be achieved after acute hypoxia, while sequelae or even dementia can be observed after chronic hypoxia, perhaps due to the different molecular mechanisms. Cardiopulmonary compensatory response, glycolysis, oxidative stress, calcium overload, adenosine, mitochondrial disruption, inflammation and excitotoxicity contribute to the molecular mechanisms of cognitive deficit after acute hypoxia. During the chronic stage of hypoxia, different adaptive responses, impaired neurovascular coupling, apoptosis, transcription factors-mediated inflammation, as well as Aß accumulation and tau phosphorylation account for the neurocognitive deficit. Moreover, brain structural changes with hippocampus and cortex atrophy, ventricle enlargement, senile plaque and neurofibrillary tangle deposition can be observed under chronic hypoxia rather than acute hypoxia.

Extraction optimization, physicochemical property, antioxidant activity, and α-glucosidase inhibitory effect of polysaccharides from lotus seedpods.

BACKGROUND: Lotus seedpods are an agricultural by-product of lotus (Nelumbo nucifera Gaertn.), which is widely cultivated in Southeast Asia and Australia. Most lotus seedpods are considered waste and are abandoned or incinerated, resulting in significant waste of resources and heavy environmental pollution. For recycling lotus seedpods, the extraction optimization, physicochemical properties, antioxidant activity, and α-glucosidase inhibitory effect of the polysaccharides contained therein were investigated in this study. RESULTS: Hot water extraction of lotus seedpod polysaccharides was optimized by using a response surface methodology combined with a Box-Behnken design, with the optimum conditions being as follows: a liquid/solid ratio of 25.0 mL g-1 , an extraction temperature of 98.0 °C, and an extraction time of 138.0 min. Under these conditions, an experimental yield of 5.88 ± 0.06% was obtained. Physicochemical analyses suggested that lotus seedpod polysaccharides belong to acidic heteropolysaccharides and are principally composed of rhamnose, arabinose, galactose, glucose, mannose, and galacturonic acid. The polysaccharides content has a broad molecular weight distribution (2.15 × 105 to 1.77 × 107 Da), an α-configuration, and mainly possesses smooth and sheet-like structures. Biological evaluations showed that the polysaccharides possessed good scavenging activity on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 1,1-diphenyl-2-picryl-hydrozyl, and hydroxyl radicals, and exerted an obvious inhibitory effect on α-glucosidase activity. Moreover, the polysaccharides content was determined to be a mixed-type noncompetitive inhibitor of α-glucosidase. CONCLUSION: The results indicate that lotus seedpod polysaccharides have potential as natural antioxidants and hypoglycaemic substitutes. This study provides the theoretical bases for the exploitation and application of polysaccharides from lotus seedpod by-product resources. © 2022 Society of Chemical Industry.

Construction of an expression vector mediated by the dual promoter for prokaryotic and mammalian cell expression system.

The aim of this study was to construct an expression vector mediated by the dual promoter that can simultaneously drive the recombinant protein production in eukaryotic and prokaryotic cells. The prokaryotic T7 promoter and ribosome binding site (RBS) was cloned downstream of CMV promoter in the eukaryotic expression vector pIRES-neo, and T7 termination sequence was inserted upstream of neomycin phosphotransferase gene to generate the dual promoter vector. The enhanced green fluorescent protein (eGFP) gene was used as reporter gene. Then, the resultant vector was transfected into Chinese hamster ovary (CHO) cells and transformed into Escherichia coli (E. coli) BL21, and the eGFP expression levels were analyzed by fluorescence microscopy, flow cytometry and Western blot, respectively. Fluorescence microscopy revealed that the eGFP was expressed in both CHO cells and E. coli BL21. Flow cytometry showed that the eGFP expression level had no significant difference between the dual promoter vector and control vector in transfected CHO cells. Western blot analysis indicated the eGFP expressed in transformed E. coli. In conclusion, a prokaryotic-eukaryotic double expression vector was successfully constructed, which has potential applications in rapid cloning and expression of recombinant proteins in both prokaryotic and eukaryotic expression systems.

FBXW7γ is a tumor-suppressive and prognosis-related FBXW7 transcript isoform in ovarian serous cystadenocarcinoma.

Aim: To explore FBXW7 protein-coding transcript isoform (α, ß and γ) expression, their functions and prognostic value in ovarian serous cystadenocarcinoma (OSC). Materials & methods: FBXW7 transcript data were collected from The Cancer Genome Atlas and the Genotype-Tissue Expression project. IOSE, A2780 and SKOV3 cells were used for in vitro and in vivo studies. Results: FBXW7α and FBXW7γ are dominant protein-coding transcripts that were downregulated in OSC. FBXW7γ overexpression reduced the protein expression of c-Myc, Notch1 and Yap1 and suppressed OSC cell growth in vitro and in vivo. FBXW7γ expression was an independent indicator of longer disease-specific survival (HR: 0.588; 95% CI: 0.449-0.770) and progression-free survival (HR: 0.708; 95% CI: 0.562-0.892). Conclusion: FBXW7γ is a tumor-suppressive and might be the only prognosis-related FBXW7 transcript in OSC.

Distance effect characteristic of the matrix attachment region increases recombinant protein expression in Chinese hamster ovary cells.

OBJECTIVES: Previously, we have found that the matrix attachment region (MAR) may confer a 'distance effect' on transgene expression. This work aims to systematically explore the increased transgene expression in transfected Chinese hamster ovary (CHO) cells due to the characteristics of MAR and its mechanism. RESULTS: Compared with the control vector, 500 and 1000 bp DNA distances between MAR and the cytomegalovirus promoter can increase transgene expression by 1.77- and 1.56-fold, respectively. Meanwhile, transgene expression was not affected when 2000 and 2500 bp spacer DNAs were inserted, but a declining trend was observed when a 1500 bp spacer DNA was inserted. The vector containing a 500 bp DNA distance significantly increased the expression of the enhanced green fluorescent protein, and this increase was not related to transgene copy numbers. CONCLUSIONS: A short DNA distance-containing MAR confers high transgene expression level in transfected CHO cells, but a distance threshold does not exist in the vector system.

Novel short synthetic matrix attachment region for enhancing transgenic expression in recombinant Chinese hamster ovary cells.

Matrix attachment regions (MARs) are DNA fragments with specific motifs that enhance transgenic expression; however, the characteristics and functions of these elements remain unclear. In this study, we designed and synthesized three short chimeric MARs, namely, SM4, SM5, and SM6, with different numbers and orders of motifs on the basis of the features and motifs of previously reported MARs, namely, SM1, SM2, and SM3, respectively. Expression vectors with six synthetic MARs flanking the down or upstream of the expression cassette for enhanced green fluorescence protein (EGFP) were constructed and introduced into Chinese hamster ovary (CHO) cells. Results indicated that the EGFP expression of the CHO cells with transfection bySM4, SM5, or SM6-containing vectors was higher than that of those containing SM1, SM2, or SM3 regardless of the MAR insertion position. The improving effect of SM5 was particularly pronounced. Transgenic expression was further enhanced with the increasing SM5 copy number. Bioinformatics analysis indicated that several arrangements of the DNA-binding motifs for CEBP, FAST, Hox, glutathione, and NMP4 may help increase transgenic expression levels and the average population of highly expressed cells. Our findings on novel synthetic MARs will help establish stable expression systems in mammalian cells.

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells.

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2'-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.

Recent developments in Hericium erinaceus polysaccharides: extraction, purification, structural characteristics and biological activities.

Hericium erinaceus (H. erinaceus), an edible mushroom with medicinal value, has a long history of usage in China and other oriental countries. Polysaccharide is supposed to be one of the major bioactive compounds in H. erinaceus, which possesses immunomodulating, anti-cancer, antioxidant, gastroprotection and intestinal health promotion, neuroprotective, hepatoprotective, antihpyerglycemic and hypolipidemic activities. In this review, the current advancements on extraction, purification, structural characteristics and biological activities of polysaccharide from different sources (fruiting body, mycelium and culture broth) of H. erinaceus were summarized. Among these aspects, summaries of the structural characteristics focused on the purified polysaccharides. Meanwhile, comparisons on the structural characteristics among the purified polysaccharides obtained from above three sources were made. Moreover, their biological activities were introduced on the basis of in vivo and in vitro experiments, and some possible action mechanisms were listed. Furthermore, the structure-activity relationship of the polysaccharide was discussed. New perspectives for the future work of Hericium erinaceus polysaccharide were also proposed. HIGHLIGHTS Extraction, purification, structural characteristics and biological activities of Hericium erinaceus polysaccharide (HEP) were summarized. Structural characteristics of the purified polysaccharides from different sources (fruiting body, mycelium and culture broth) of Hericium erinaceus were summarized and compared. Structure-activity relationship of HEP was discussed, and new perspectives for the future work of this polysaccharide were proposed.

Age-Related Impaired Efficacy of Bone Marrow Cell Therapy for Myocardial Infarction Reflects a Decrease in B Lymphocytes.

Treatment of myocardial infarction (MI) with bone marrow cells (BMCs) improves post-MI cardiac function in rodents. However, clinical trials of BMC therapy have been less effective. While most rodent experiments use young healthy donors, patients undergoing autologous cell therapy are older and post-MI. We previously demonstrated that BMCs from aged and post-MI donor mice are therapeutically impaired, and that donor MI induces inflammatory changes in BMC composition including reduced levels of B lymphocytes. Here, we hypothesized that B cell alterations in bone marrow account for the reduced therapeutic potential of post-MI and aged donor BMCs. Injection of BMCs from increasingly aged donor mice resulted in progressively poorer cardiac function and larger infarct size. Flow cytometry revealed fewer B cells in aged donor bone marrow. Therapeutic efficacy of young healthy donor BMCs was reduced by depletion of B cells. Implantation of intact or lysed B cells improved cardiac function, whereas intact or lysed T cells provided only minor benefit. We conclude that B cells play an important paracrine role in effective BMC therapy for MI. Reduction of bone marrow B cells because of age or MI may partially explain why clinical autologous cell therapy has not matched the success of rodent experiments.

SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF-1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. qRT-PCR and flow cytometry were used to explore eGFP expression levels. And gene copy number was also detected by qPCR, respectively. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in CHO cells.

CRISPR/Cas9-mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long-term stability.

CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a-deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a-deficent CHO cell line based on Dnmt3a KO displayed an enhanced long-term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a-deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a-deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.