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Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability.

BackgroundAs the demand for monoclonal antibodies (mAb) increases, more efficient expression methods are required for their manufacturing process. Transcriptional gene silencing is a common phenomenon in recombinant cell lines which leads to expression reduction and instability. There are reports on improved antibody expression in ubiquitous chromatin opening element (UCOE) containing both heavy and light chain gene constructs. Here we investigate the impact of having these elements as part of the light chain, heavy chain or both genes during cell line development. In this regard, non-UCOE and UCOE vectors were constructed and stable Chinese hamster ovary (CHO) cell pools were generated by different vector combinations.ResultsExpression analysis revealed that all UCOE cell pools had higher antibody yields compared to non-UCOE cells, Moreover the most optimal expression was obtained by cells containing just the UCOE on heavy chain. In terms of stability, it was shown that the high level of expression was kept consistence for more than four months in these cells whereas the expression titers were reduced in the other UCOE pools.ConclusionsIn conclusion, UCOE significantly enhanced the level and stability of antibody expression and the use of this element with heavy chain provided more stable cell lines with higher production level.

Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site-specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO-S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570-1576, 2016.

Monoclonal antibodies (mAbs) are crucial in pharmaceutical biotechnology. Mammalian cell lines are the most preferred for their production. One of the significant challenging issues of mammalian expression systems is epigenetic gene silencing. Employing epigenetic gene regulation tools can increase the productivity of the mammalian cell lines. Sodium butyrate (NaBut) and valproic acid (VPA) regulate gene expression by inhibiting histone deacetylase. A ubiquitous chromatin-opening element (UCOE) can improve expression by reducing DNA methylation. Here, the separate and combined effects of NaBut and VPA histone deacetylase inhibitors (iHDACs) and UCOE on mAb synthesis were studied. Stable cell lines were generated by non-UCOE (CHO-HL) and UCOE-containing vectors (CHO-UHUL) and cultured in the presence and absence of NaBut or VPA. Expression analysis showed that CHO-UHUL gave a 4-fold greater yield than non-UCOE CHO-HL. Antibody production levels of the CHO-HL and CHO-UHUL cells increased 2-fold and 2.5-fold after NaBut and VPA treatment, respectively. These results indicate that UCOE has more impact on antibody expression than iHDACs. iHDAC treatment exhibited at least a 0.5-fold higher antibody yield in UCOE containing CHO-UHUL cells. Thus utilization of NaBut and VPA (iHDACs) and UCOE resulted in antibody expression improvement, and the combined use of them had a synergistic effect on antibody synthesis.

Purpose Modern construction methods have been developed with the goal of reducing construction time as much as possible, which results in some situations during construction and within the first few days after it, when concrete is subjected to exceptionally high loads. The precast concrete, which is the concrete in very early ages, may result in severe cracks or damages. In conventional construction projects, sometimes working with concrete, which had not reached its ultimate strength, is an unavoidable matter of fact. This paper aims to discuss these issues. Design/methodology/approach Researchers in the field of construction materials have done their best to make some changes in the different parts of the concrete in order to bring about reforms, based on the existing needs, and achieve new quality and primacy from concrete. One kind of concrete, the emergence of which dates back to many years ago, is self-compacting concrete. Thanks to its high efficiency for the parts with complex forms of high-density steel, this kind of concrete suggests new prospects. Findings This study aims at evaluating the effect of early loads on the 28-day compressive strength of concretes with zeolite and limestone powder under different curing conditions (wet or dry). In this regard, two self-compacting concrete mix designs with the same ratio of water to cementations materials and 0.4 percent and 10 percent zeolite have been considered; therefore, concrete cube samples with zeolite and limestone powder in different curing conditions at ages of three, one and seven days under preloading with 80–90 percent of compressive strength are damaged, and after curing in different conditions, their 28-day compressive strength is measured. According to the results, the recovery of the 28-day compressive strength of damaged samples, compared to that of intact samples, is possible in all curing conditions. The experiments that have been performed on concrete samples under dry and wet curing conditions show that the full recovery of compressive strength of damaged samples compared to that of intact ones happened only in preloaded samples at the age of one days, and in other ages (three and seven days) the 28-day strength reduction has occurred in damaged samples compared to the that in intact samples. The results of concrete samples with zeolite and without limestone powder at the age of one day indicate the greatest impact on other samples on the 28-day compressive strength of damaged samples compared to that of intact ones, occurring under dry condition. Originality/value This research analyzed and studied the influence under wet and dry curing conditions and the presence of limestone powder and zeolite fillers in recovering of the 28-day compressive strength of preloaded concrete samples at early stages (one, three and seven days) after the construction of the concrete.

Background:Nowadays, owing to medicinal plants as a candidate to obtain promising new medicinal agents, there is a renewed interest in the use of these natural sources for drug development.Objective:In the present study, we aimed to assess the anticholinesterase, antioxidant, and neuropotective effects of Tripleurospermum disciforme and Dracocephalum multicaule extracts.Materials and Methods:Methanolic extract of the plants was prepared by maceration method. Anticholinesterase effect of different concentrations of the plants was studied by colorimetric method and antioxidant activity was evaluated using diphenypicrylhydrazil (DPPH) assay. Protective effect of the extracts against amyloid β (Aβ)-induced toxicity in PC12 cells was determined by MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method.Results:Both T. disciforme and D. multicaule extracts could inhibit acetylcholinesterase (AChE) in a dose-dependent manner. The highest inhibition occurred at 5 μg/ml (71.18 ± 4.9 and 79.06 ± 3.1% inhibition respectively by T. disciforme and D. multicaule) in comparison to tacrine (86.37 ± 3.24%). The greatest DPPH inhibition of T. disciforme and D. multicaule was shown at 800 μg/ml (89.04 ± 3.9 and 78.5 ± 3.7%, respectively). None of tested extracts induced protection against βA toxicity in PC12 cell.Conclusion:Although the results indicated anticholinesterase and antioxidant of the T. disciforme and D. multicaule, further specific studies and scientific validity are needed.