Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells.

In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed.

Fusion with matrix attachment regions enhances expression of recombinant protein in human HT-1080 cells.

Like endogenous proteins, recombinant foreign proteins produced in human cell lines also need post-translational modifications. However, high and long-term expression of a gene of interest (GOI) presents significant challenges for recombinant protein production in human cells. In this work, the effect of human matrix attachment region elements (MARs), including the ß-globin MAR (gMAR), chicken lysozyme MAR (cMAR), and a combination of these two, on the stable expression of GOI was assessed in human HT-1080 cells. After transfection with vectors containing the MAR elements and eGFP, stably HT-1080 cell pools were obtained under selective pressure. eGFP protein expression was analyzed by flow cytometry, while transgene copy number and eGFP mRNA expression levels were determined with qPCR and qRT-PCR technology. We found that MARs could not enhance transfection efficiency, but gMAR could significantly increase eGFP expression in stable HT-1080 cell pools by approximately 2.69-fold. Moreover, gMAR could also increase eGFP expression stability during long-term culture. Lastly, we showed that the effect of the MARs on transgenes was related to the gene copy number. In summary, this study found that MARs could both enhance the transgene expression and stability in HT-1080 cells.

CYP3A4 and microRNA-122 are involved in the apoptosis of HepG2 cells induced by ILs 1-decyl-3-methylimidazolium bromide.

Ionic liquids (ILs) as green alternatives for volatile organic solvents are increasingly used in commercial applications. It is necessary to explore the cytotoxic mechanism of ILs to reduce the risk to human health. For this purpose, cell viability, apoptosis, cytochrome P450 3A4 (CYP3A4), glucose transporter type 2 (GLUT2), and microRNA-122 (miR-122) gene expression in HepG2 cells was evaluated after IL exposure. The results showed that ILs reduced the viability of HepG2 cells through apoptotic cell death. Moreover, ILs markedly upregulated the transcription and protein levels of CYP3A4, but did not affect the expression of GLUT2 in either messenger RNA level or protein level. Finally, ILs increased the expression of miR-122 and inhibition of miR-122 with miR-122 inhibitor blocked ILs-induced apoptosis in HepG2 cells. This finding may contribute to an increased understanding of the in vitro molecular toxicity mechanism of ILs to further understand IL-related human health risks.

Retraction Note: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.

This paper has been retracted at the request of the authors.

Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Toxic cytological alteration and mitochondrial dysfunction in PC12 cells induced by 1-octyl-3-methylimidazolium chloride.

Ionic liquids have recently received considerable attention due to their negligible vapor pressure and substitute for conventional organic solvents. However, their solubility in water and a great deal of literature regarding their toxicity on aquatic organisms have caused much concern in recent years. This study aims to evaluate the cytotoxicity of 1-octyl-3-methylimidazolium chloride ([C(8)mim][Cl]) on the rat pheochromocytoma (PC12) cell line by cell viability assay and to determine the cytological alterations and damages in PC12 cells after 24h of exposure at the concentrations of 0.07, 0.14, and 0.28 mM of [C(8)mim][Cl]. The results show that [C(8)mim][Cl] inhibits PC12 cell growth and decreases their viabilities in a remarkable dose-dependent manner, and the 24h EC(50) of [C(8)mim][Cl] for PC12 cells is calculated to be around 0.56 mM. Our results also reveal that [C(8)mim][Cl]-exposure induces DNA damage, sustained increase of intracellular Ca(2+), overproduction of reactive oxygen species, gradually exhausted cellular ATP, mitochondrial permeability transition, and apoptosis in PC12 cells. We suppose that mitochondrial permeability transition and mitochondrial dysfunction maybe the major cytotoxicity mechanism of [C(8)mim]Cl for PC12 cells.

Apoptosis caused by imidazolium-based ionic liquids in PC12 cells.

The cytotoxicity of alkylmethylimidazolium-based ionic liquids on rat pheochromocytoma (PC12) cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release (LDH), and acridine orange staining in the present study. Mitochondrial depolarization, DNA fragmentation, reactive oxygen species (ROS) levels, and caspase-3 activity were also determined. The results showed dose-dependent cytotoxicity of ionic liquids on PC12 cells, and the ionic liquids with longer lateral chains had stronger cytotoxicity. Additionally, we found that exposure to the ionic liquid 1-octyl-3-methylimidazolium bromide ([C(8)mim]Br) provoked cellular LDH release, increased mitochondrial depolarization, induced cellular transmogrification, nuclear shrinkage and DNA fragmentation, and promoted caspase-3 activity and ROS levels in PC12 cells. These results suggest that [C(8)mim]Br may induce PC12 cell apoptosis triggered by excessive ROS and mediated by mitochondrial depolarization and permeability transition. Our result may be helpful for illuminating the cytotoxicity mechanism of alkylmethylimidazolium-based ionic liquids and safely using them in the future.

Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells.

Previous work has shown that the MAR (matrix attachment region) could increase transgene expression in stably transfected CHO (Chinese-hamster ovary) cells. To study the positional effect of MAR on transgene expression, three expression vectors were constructed which contained the human beta-globin MAR in different sites, including the vector with two MARs flanking the CAT (chloramphenicol acetyltransferase) expression cassette, one MAR at the 59 or 39 site. These vectors were transfected into CHO cells. The level of CAT gene expression was most effectively increased by two MARs flanking the CAT expression cassette. This increase was also seen when MAR was inserted at the 59 site upstream of the expression cassette, whereas the transgene expression level decreased when MAR was inserted at the 39 site downstream of the expression cassette. We have also shown that the transgene expression level is not directly proportional to the gene copy number, and gene copy number dependency does not exist.

Nucleotide sequence and expression of the 14-3-3 from the halotolerant alga Dunaliella salina.

Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Dunaliella salina, however, the nucleotide sequence of this gene have not been reported so far. In the present study, the cloning and characterization of the nucleotide sequence, the gene copy and expression were undertaken. The coding sequence of the gene was found to be interrupted by five introns of 132, 266, 153, 152 and 625 bp, respectively. Introns 3-5 were found in conserved positions as compared to the Chlamydomonas reinhardtii 14-3-3 gene. D. salina 14-3-3 cDNA was inserted into the prokaryotic expression plasmid pET-28 and transformed into E. coli BL21, and the recombinant expressed 14-3-3 protein was purified from E. coli and immunized the rabbit. Indirect ELISA coated with 14-3-3 illustrated that the rabbit antisera titration was 1:1.00E + 06. Western blotting assays confirmed that prepared rabbit antibodies could recognize the recombinant 14-3-3 protein. Southern blotting results showed that there was only one copy of the 14-3-3 present in the genome of D. salina and 14-3-3 expression did not change throughout the Dnualiella cell cycle.