Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells.

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells.

Recombinant antibodies are rapidly developing therapeutic agents; approximately 40 novel antibody molecules enter clinical trials each year, most of which are produced from Chinese hamster ovary (CHO) cells. However, one of the major bottlenecks restricting the development of antibody drugs is how to perform high-level expression and production of recombinant antibodies. The high-efficiency expression and quality of recombinant antibodies in CHO cells is determined by multiple factors. This review provides a comprehensive overview of several state-of-the-art approaches, such as optimization of gene sequence of antibody, construction and optimization of high-efficiency expression vector, using antibody expression system, transformation of host cell lines, and glycosylation modification. Finally, the authors discuss the potential of large-scale production of recombinant antibodies and development of culture processes for biopharmaceutical manufacturing in the future.

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.

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.

Effect of promoter, promoter mutation and enhancer on transgene expression mediated by episomal vectors in transfected HEK293, Chang liver and primary cells.

The episomal vector cannot integrate into the host cell chromosome, which has no potential risk in gene therapy. However, the low level of transgene expression driven by episomal vectors needs to be solved. In this study, we investigated the effects of enhancers, promoters and promoter variants on transgene expression levels driven by episomal vectors in HEK293, Chang liver and primary cells. Results showed that all eight cis-acting elements used could increase transfection efficiency and transient eGFP expression in transfected HEK293 and Chang liver cells. In stably transfected mammalian cells, the elongation factor-1 alpha (EF-1α) promoter and mutant-404 showed high and stable transgene expression. The mechanisms might be related to the type and quantity of transcription factor regulatory elements. Moreover, quantitative reverse transcription polymerase chain reaction analysis showed that mRNA expression levels were not directly proportional to protein expression levels. Furthermore, the EF-1α promoter conferred high transgene expression levels in primary cells, and the plasmid was also present in the episomal state. Taken together, these results provided valuable information for improving transgene expression with episomal vectors in mammalian cells.

Lentiviral-Mediated Overexpression of MicroRNA-141 Promotes Cell Proliferation and Inhibits Apoptosis in Human Esophageal Squamous Cell Carcinoma.

BACKGROUND: Esophageal Carcinoma (EC) is the eighth most common cancer worldwide. Numerous studies have highlighted a vital role of microRNAs (miRNAs) in the development of EC. However, the mechanism of microRNA (miRNA)-141 in Esophageal Squamous Cell Carcinoma (ESCC) remains unknown. OBJECTIVE: In this study, we explored the effects of miRNA-141 on EC cell proliferation, apoptosis, xenograft tumour growth and their possible mechanisms. METHODS: A lentivirus-vector-expressing miRNA-141 was constructed, and a TE-1 cell line of ESCC with a stable expression of miRNA-141 was transfected and screened. The miRNA-141 expression level was detected using qRT-PCR. Effects of miRNA-141 overexpression on cell proliferation and apoptosis were detected using MTT and flow cytometry, respectively. Using a dual-luciferase reporter assay, a direct interaction between miRNA-141 and the 3'-Untranslated Region (UTR) of YAP1 and SOX17 was confirmed. Tumour xenograft experiment in nude mice was used to detect the tumour growth, and the effects of miRNA-141 overexpression on YAP1 and SOX17 were analysed using Western blot. RESULTS: We found that miRNA-141 was highly expressed in TE-1 cells, and miRNA-141 overexpression promoted cell proliferation and inhibited apoptosis. Moreover, the miRNA-141 group showed significantly increased tumour growth ability, luciferase activities and expression levels of YAP1 and SOX17 in the miRNA-141group were significantly down-regulated. CONCLUSION: miRNA-141 promotes cell proliferation and inhibits apoptosis in ESCC by downregulating the expression level of YAP1 and SOX17, indicating that miRNA-141 may be a potential molecular target for the treatment of ESCC.

A Simple, Time-Saving Dye Staining of Proteins in Sodium Dodecyl Sulfate-Polyacrylamide Gel Using Coomassie Blue.

Most traditional post-electrophoretic processes need several hours to several days to finish the whole staining process and traditional staining solutions all contain methanol, acetic acid, or phosphoric acid, which not only produce the unpleasant smell but also cause environmental pollution. Here a fixation-free, fast protein staining method in sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol includes only staining and quick washing steps, can be completed in 0.5 h. It has a sensitivity of 10 ng. In addition, the dye stain does not contain any acid or methanol.

[Effects of Different Promoters and MAR Combinations on Transgene Expression of Recombinant CHO Cells].

OBJECTIVE: To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells. METHODS: The expression vector was constructed by the combination of beta globin MAR (gMAR) with the human cytomegalovirus immediate-early promoter (CMV-IE) and simian virus 40 (SV40) promoter. These vectors were transfected into CHO cells,after 48 h,the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines,and the expression level of eGFP in CHO cells was analyzed by flow cytometry. The relative copy numbers of eGFP were analyzed by qPCR. RESULTS: Without gMAR expression vector,the expression of eGFP which was driven by CMV-IE promoter was stronger than that of SV40 promoter; gMAR could increase the expression level of eGFP driven by CMV-IE promoter,but did not show any enhancement in SV40 promoter. The expression level of eGFP which containing gMAR on both sides was stronger than that of gMAR on one side driven by CMV-IE promoter; After G418 screening,the expression level of eGFP containing gMAR driven by SV40 promoter wasunstable,the fluorescence gradually weakened,therefore,we only analyzed the expression vector stably expressing the eGFP gene driven by CMV-IE promoter by flow cytometry and qPCR. Compared with the expression vector without gMAR containing CMV-IE promoter,flow cytometry showed that the expression levels of eGFP on one and both sides with gMAR were increased by 9.85-fold and 12.94-fold,respectivley; The result of qPCR showed that the copy number of the eGFP gene without gMAR was set to 1,the copy number of the eGFP gene in the expression vector driven by CMV-IE with gMAR on one side and both sides were 3.68-fold and 9.25-fold,respectively. CONCLUSION: The activity of CMV-IE promoter is stronger than that of SV40 promoter. gMAR can enhance the expression levels of transgene,which may be related to the increase of gene copy number.

Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.

Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human ß-interferon and ß-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.

Cell Compatibility of an Eposimal Vector Mediated by the Characteristic Motifs of Matrix Attachment Regions.

The characteristic sequence of ß-interferon matrix attachment regions (MARs) can mediate transgene expression via episomal vectors in Chinese hamster ovary (CHO) cells. However, the host cells were from hamster ovaries, which are not suitable target cells for gene therapy. In this study, we aimed to evaluate the suitability of 12 different human cell lines as target cells for gene therapy. We transfected the cells with episomal vectors and obtained colonies stably expressing the vector products after G418 screening. Therefore the stably transfected cells were split into two and further cultured either in the presence or the absence of G418. Flow cytometry was used to observe the positive rate of cell transfection and level of green fluorescent protein (GFP) expression. Plasmid rescue assays, fluorescence in situ hybridization (FISH), and fluorescence quantitative polymerase chain reaction (PCR) were used to investigate the presence and gene copy numbers of plasmid in mammalian cells. The results showed that transfection efficiency and transgene expression levels in A375, Eca-109, and Changliver cells were high. In contrast, transgene silencing was observed in BJ, HSF, and A431 cells, and low expression of the transgene was observed in the other six cell lines. In addition, the plasmid was present in the episomal state in A375, Eca-109, and Chang-liver cells with relatively low copy numbers even under nonselective conditions. Thus, our results provide the first evidence showing transgene expression of an episomal vector mediated by the characteristic motifs of MARs for maintenance of the longterm stability of episomes in different types of cells.

Episomal Lentiviral Vector-Mediated miR-145 Overexpression Inhibits Proliferation and Induces Apoptosis of Human Esophageal Carcinomas Cells.

BACKGROUND: Gene therapy is a promising approach for the treatment of various cancers. However, most viral vectors used for this purpose carry risks, including potential integration into the host genome. OBJECTIVE: We addressed this issue in the present study by constructing an episomal lentiviral vector using the .-interferon matrix attachment region to express the microRNA -145(miR-145), and examining the effect of miR-145 overexpression on human esophageal carcinomas (EC) cells. Some recent relevant patents are also discussed. METHOD: Expression levels of miR-145 and the marker protein enhanced green fluorescent protein (EGFP) in infected ECA109 and EC9706 human esophageal carcinoma cells were detected by quantitative PCR and flow cytometry, respectively. Cell proliferation and apoptosis were assessed by Cell Counting Kit-8 and flow cytometry, respectively. Plasmid rescue experiments and fluorescence in situ hybridization were used to determine the episomal status of the transfected vector. RESULTS: We found that EGFP and miR-145 were highly expressed in EC cells, and miR-145 overexpression inhibited cell proliferation and induced apoptosis. Moreover, the lentiviral vector did not integrate into the host genome, but was maintained episomally at lower copy numbers. CONCLUSION: Taken together, our results demonstrate that miR-145-expressing episomal lentiviral vectors are a promising tool for gene therapy in the treatment of EC.

Impact of Different Promoters on Episomal Vectors Harbouring Characteristic Motifs of Matrix Attachment Regions.

We previously demonstrated that the characteristic sequence of matrix attachment regions (MARs) allows transgenes to be maintained episomally in CHO cells. In the present study, six commonly used promoters from human cytomegalovirus major immediate-early (CMV), simian vacuolating virus 40 (SV40), Rous sarcoma virus, Homo sapiens ubiquitin C, phosphoglycerate kinase, and ß-globin, respectively, were evaluated to determine their effects on transgene expression and stability in CHO cells stably transfected via the episomal vector harbouring characteristic MAR motifs. The CHO cells were transfected with vectors and then screened using G418, after which the stably transfected cells were split into two and further cultured either in the presence or absence of G418. Of the six promoters, the CMV promoter yielded the highest transgene expression levels and the highest transfection efficiency, whereas the SV40 promoter maintained transgene expression more stably during long-term culture than the other promoters did. The CMV and SV40 promoter-containing vectors were furthermore episomally maintained and conferred sustained eGFP expression in the cells even under nonselective conditions. On the basis of these findings, we conclude that the CMV promoter performs best in terms of yielding both high expression levels and high levels of stability using this episomal vector system.

Treating Colon Cancer Cells with FK228 Reveals a Link between Histone Lysine Acetylation and Extensive Changes in the Cellular Proteome.

The therapeutic value of FK228 as a cancer treatment option is well known, and various types of cancer have been shown to respond to this drug. However, the complete mechanism of FK228 and the affect it has on histone lysine acetylation and the colon cancer cell proteome are largely unknown. In the present study, we used stable isotope labeling by amino acids in cell culture (SILAC) and affinity enrichment followed by high-resolution liquid chromatograph-mass spectrometer (LC-MS)/MS analysis to quantitate the changes in the lysine acetylome in HCT-8 cells after FK228 treatment. A total of 1,194 lysine acetylation sites in 751 proteins were quantified, with 115 of the sites in 85 proteins being significantly upregulated and 38 of the sites in 32 proteins being significantly downregulated in response to FK228 treatment. Interestingly, 47 histone lysine acetylation sites were identified in the core histone proteins. We also found a novel lysine acetylation site on H2BK121. These significantly altered proteins are involved in multiple biological functions as well as a myriad of metabolic and enzyme-regulated pathways. Taken together, the link between FK228 function and the downstream changes in the HCT-8 cell proteome observed in response to FK228 treatment is established.

Distance effect of matrix attachment regions on transgene expression in stably transfected Chinese hamster ovary cells.

The ß-globin matrix attachment regions (MARs) were inserted into the 5'-site of the eukaryotic expression vector cassette and DNA fragments 350 and 750 bp in length were inserted into the site to generate expression vectors with varying distances between the expression cassette and MAR. The vectors containing MARs increased chloramphenicol acetyltransferase (CAT) expression levels compared to the negative control vector lacking the MAR; the highest expression increase was 3.8-fold. A greater MAR-transgene distance (750 bp) correlated with a greater increase in transgene expression when compared to the control vector that lacked separation between the MAR and transgene. CAT gene copy numbers were higher in cells transformed with the vector possessing a smaller MAR-transgene distance (350 bp) than in cells belonging to the other three groups. However, MAR-induced transgene expression levels did not exhibit a direct relationship with gene copy number.

Establishment of an optimized PCR method using sequence-specific primers for screening multiple gene polymorphisms simultaneously.

The present study aimed to explore the factors that affect polymerase chain reaction using sequence-specific primers (PCR-SSP) and to establish an optimized PCR-SSP method for detecting multiple gene polymorphisms simultaneously. The amplification system parameters, including the concentrations of Mg2+, dNTPs, pfu Taq, primers and control primers, were optimized using the designed PCR-SSP reactions. The resulting optimized reaction system was used to determine the melting temperature of the genomic DNA and the cycling parameters. The optimized PCR-SSP method was used to analyze the polymorphisms of the following genes: mutations -308A/G and -238G/A in TNF-α, -174G/C in IL-6 and C/T mutation at exon 188 of CYP2D6 *10B. The PCR-SSP amplification system was optimized; in a 20 µl reaction system, the quantities of Mg2+, dNTPs, pfu Taq, primers, control primers and genomic DNA were 3.25 µM, 0.5 mM, 2.5 units, 0.5 µM, 0.2 µM and 0.15 µg, respectively. The cycling system comprised 5 start cycles and took 15 min to melt a genomic DNA sample using a touchdown protocol. The optimized PCR-SSP is suitable for polymorphism analysis of polygenic SNPs in large genomic DNA samples and a number of different genes.

A simple and practical method that prepares high molecular weight DNA ladders.

The purpose of the current study was to report a simple and practical method to prepare high molecular weight (mw) DNA ladders. The method involves 1,000-4,000-base pairs (bp) DNA fragments being amplified by polymerase chain reaction (PCR), using λ DNA as a template. The constructed plasmids are digested by restriction endonucleases to produce 5-, 6-, 8- and 10-kb DNA fragments, followed by purification and precipitation with ethanol, and mixed proportionally. The 1,000-4,000-bp DNA fragments were successfully generated by PCR and 5-, 6-, 8- and 10-kb DNA fragments were obtained through the digestion of the plasmids. The bands of the prepared high mw DNA ladder were clear and may aid future molecular biology studies.

Different matrix attachment regions flanking a transgene effectively enhance gene expression in stably transfected Chinese hamster ovary cells.

Numerous matrix attachment regions (MARs) have been used to improve transgene expression in genetic engineering, but an efficient and stable expression vector is lacking. In the present study, a vector named pCCF containing chloramphenicol acetyltransferase (CAT) reporter gene cassettes was constructed. The cassettes were flanked by a ß-interferon MAR at the 5' upstream of the reporter gene cassettes, and a ß-globin MAR at the 3' site. After transfecting pCCF into Chinese hamster ovary cells, the expression level of the CAT gene with a MAR was effectively increased to about 4.5-fold higher than that transfected with pCAM (containing two ß-globin MARs flanking the expression cassette), and to 46.4-fold higher than that transfected with the control plasmid pCAG (without MARs). Quantitative reverse transcription polymerase chain reaction and the 2(-ΔΔCt) method were used to analyze the CAT gene relative copy numbers. The expression levels were found to be not directly proportional to the gene copy numbers when MAR elements from different sources were used. However, the presence of MARs improved the transgene copy numbers.

A simple and effective SuperBuffer for DNA agarose electrophoresis.

In the paper, we describe a unique effective electrophoresis buffer for DNA agarose electrophoresis, called SuperBuffer. Using this buffer, electrophoresis could be performed within 10 min at voltages as high as 25V/cm. In addition, DNA fragments of different lengths could be isolated clearly even at lower agarose gel concentrations and the DNA recovery efficiency was higher than that of the TAE/TBE running buffers. The SuperBuffer still retained its electrophoretic effect even after several uses.

Simple, time-saving dye staining of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue.

A fixation-free and fast protein-staining method for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye stain does not contain any amount of acid and methanol, such as phosphoric acid. Considering the speed, simplicity, and low cost, the dye stain may be of more practical value than other dye-based protein stains in routine proteomic research.

[Infection and physico-chemical characteristics of Anisakis among marine fish caught in Zhoushan Fishery].

OBJECTIVE: To study the rates of infection and physicochemical characteristics of the third stage Anisakis simplex larvae among marine fish caught in Zhoushan Fishery. METHODS: Fish were dissected to detect Anisakis larvae and identified morphologically. The survival tolerance of the third stage Anisakis simplex larvae in various medium, anthelmintic drug, temperature were studied in laboratory. RESULTS: The total infection rate of Anisakis simplex larvae in fish was 49.10%. High rates of Anisakis infection were observed in hairtails, Pneumatophorus japonicus, Miichthys milky, Argyrosomus argentatus and Muraenesox cinereus (infection rates > 90 percent). The infection intensity of Anisakis per fish varied from 1 to 114. The mean intensity of Anisakis larvae was 15.20 per fish. 3314 Anisakis were detected in 218 marine fish. The survival tolerance of the third stage Anisakis simplex larvae in various Medium, anthelmintic drug, temperature were observed in laboratory condition. The third stage Anisakis simplex larvae showed a strong endurance to stock condiment. The anisakicidal effects of the high purity wine were more effective than that of the low purity wine. The anisakicidal effects of 6.25 g/L mebendazole composite were more effective than that of 18.75 g/L and also more effective than those of other drugs. The third stage Anisakis simplex larvae could survive with length up to 9 h and 12 h in condition of -20°C, -10°C and very sensitive to high temperature treatment. However, they could barely survive in more than 11 s and 1 s under the temperature of 50°C and 60°C. CONCLUSION: The percentage of infection was fairly high for Anisakis larvae of marine fish caught in Zhoushan Fishery. The third stage Anisakis simplex larvae was shown to have a fairly good tolerance to the external environments. The marine fish were frozen under -20°C beyond 24 h before they were sold on market and cooked with high temperature seemed to be helpful for preventing and controlling effectively the infection of Anisakis.