Studies on the influence of different designs of eukaryotic vectors on the expression of recombinant IgA

Argentova, V. V.; Алиев, Т. К.; Toporova, V. A.; Рыбченко, В. С.; Долгих, Д. А.; Kirpichnikov, Mikhaǐl P.

doi:10.3103/s0096392517020018

Cited by 7 publications

(6 citation statements)

References 15 publications

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“…In the current study, we used bipromoter and bicistronic expression plasmid constructs for the enhanced gene expression in CHO cells. Data analysis results of transient transfection in CHO cells showed that bicistronic expression plasmid design in CHO cells was better than previously reported results with bipromoter plasmid [ 8 ]. Our results indicated that compared to bipromoter expression, EMCV IRES-long link-mediated bicistronic expression constructs yield higher antibody expression levels, long-term stability, and advantage for generation of stable cell lines.…”

Section: Discussionmentioning

confidence: 66%

“…1 C and D). Two orientations of heavy chain gene cassette: head-to-tail (C) and head-to-head (D) bipromotor vectors were studied for mAb FI6-IgA production in CHO DG44 ( dhfr −/−) cells [ 8 ].…”

Section: Resultsmentioning

confidence: 99%

“…Some experiments also showed that the orientation of promoters and the presence of introns affect the expression of immunoglobulins. In addition, the optimal results were demonstrated when cells were transfected with the expression plasmid that contained introns and had a head-to-tail direction of transcription [ 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

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Optimization of recombinant antibody production based on the vector design and the level of metabolites for generation of Ig- producing stable cell lines

Toporova

Argentova

Алиев

et al. 2023

Journal of Genetic Engineering and Biotechnology

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Background The biopharmaceutical industry is significantly growing worldwide, and the Chinese hamster ovary (CHO) cells are used as a main expression host for the production of recombinant monoclonal antibodies. Various metabolic engineering approaches have been investigated to generate cell lines with improved metabolic characteristics for increasing longevity and mAb production. A novel cell culture method based on the 2-stage selection makes it possible to develop a stable cell line with high-quality mAb production. Results We have constructed several design options of mammalian expression vectors for the high production of recombinant human IgG antibodies. Versions for bipromoter and bicistronic expression plasmids different in promoter orientation and cistron arrangements were generated. The aim of the work presented here was to assess a high-throughput mAb production system that integrates the advantages of high-efficiency cloning and stable cell clones to stage strategy selection reducing the time and effort required to express therapeutic monoclonal mAbs. Development of a stable cell line using bicistronic construct with EMCV IRES-long link gave an advantage in high mAb expression and long-term stability. Two-stage selection strategies allowed the elimination of low-producer clones by using metabolic level intensity to estimate the IgG production in the early steps of selection. The practical application of the new method allows to reduce time and costs during stable cell line development.

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Section: Discussionmentioning

confidence: 66%

Section: Resultsmentioning

confidence: 99%

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Optimization of recombinant antibody production based on the vector design and the level of metabolites for generation of Ig- producing stable cell lines

Toporova

Argentova

Алиев

et al. 2023

Journal of Genetic Engineering and Biotechnology

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“…Antibodies of the IgA1 and IgA2m1 isotypes, respectively, are expressed from these plasmids. To obtain the dimeric forms of IgA, the pcDNA-J plasmid was constructed as follows; it contains the gene of the J-chain, which links monomers to form dimers, and the G418 (gentamycin) resistance gene [14,15].…”

Section: Methodsmentioning

confidence: 99%

In vitro Antiviral Activity of Recombinant Antibodies of IgG and IgA Isotypes to Hemagglutinin of the Influenza A Virus

et al. 2017

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⎯Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.

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“…We constructed a library of synthetic promoters that differed in their CpG densities. We started with a synthetic version of the human elongation factor 1α promoter [pEF1s(orig), with 18% CpG density], a 544 bp fusion of promoter fragments from the human EF1α promoter and human T-cell virus (HTLV), that is commercially available (InvivoGen) and has been used for antibody expression and gene therapies. , To identify conserved CpG elements, we compared both the EF1α fragment and HTLV fragments of this promoter to their natural orthologs, respectively . We then removed or added CG pairs into the promoter at non-conserved sites.…”

Section: Results and Discussionmentioning

confidence: 99%

Tuning Methylation-Dependent Silencing Dynamics by Synthetic Modulation of CpG Density

Budde

Zhu

et al. 2023

ACS Synth. Biol.

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Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here, we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.

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Studies on the influence of different designs of eukaryotic vectors on the expression of recombinant IgA

Cited by 7 publications

References 15 publications

Optimization of recombinant antibody production based on the vector design and the level of metabolites for generation of Ig- producing stable cell lines

Optimization of recombinant antibody production based on the vector design and the level of metabolites for generation of Ig- producing stable cell lines

In vitro Antiviral Activity of Recombinant Antibodies of IgG and IgA Isotypes to Hemagglutinin of the Influenza A Virus

Tuning Methylation-Dependent Silencing Dynamics by Synthetic Modulation of CpG Density

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