RESUMO
Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification 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 are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: ⢠The recombinant therapeutic protein degradation in CHO cell systems is reviewed. ⢠Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. ⢠Reducing the degradation can improve the quality of recombinant therapeutic proteins.
Assuntos
Apoptose , Indústrias , Animais , Cricetinae , Humanos , Células CHO , Cricetulus , ProteóliseRESUMO
Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed. KEY POINTS: ⢠Fed-batch culture in CHO cells is reviewed. ⢠Fed-batch has become a common technology for recombinant protein production. ⢠Fed batch culture promotes recombinant protein production in CHO cells.
Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Cricetinae , Animais , Humanos , Cricetulus , Células CHO , Proteínas Recombinantes/metabolismo , ImunoglobulinasRESUMO
WHAT IS KNOWN AND OBJECTIVES: Dapagliflozin was the first oral treatment approved in type 1 diabetes mellitus (T1DM) patients, simultaneously improving body weight. However, the time course and dose effect of dapagliflozin on loss of weight in T1DM patients was still unknown. The present study aimed to investigate quantitative relationship between dapagliflozin and loss of weight in T1DM patients based on Model-based Meta-analysis. METHODS: Five dapagliflozin dosage groups, two of them were 5 mg/day and three of them were 10 mg/day, 1612 T1DM patients were analysed with maximal effect (Emax ) model, and evaluation index was change rate of body weight from baseline value. RESULTS: In these T1DM patients, dosages were not incorporated into model, indicating no significant dose-response relationship between 5 and 10 mg/day affecting loss of weight. Emax and the treatment duration to reach half of the maximal effects (ET50 ) of dapagliflozin influencing loss of weight in T1DM patients were -4.9% and 10.4 weeks, and the duration to achieve 25%, 50%, 75%, and 80% (plateau) of Emax were 3.5, 10.4, 31.2, and 41.6 weeks. WHAT IS NEW AND CONCLUSIONS: It was the first time to explore quantitative relationship between dapagliflozin and loss of weight in T1DM patients. To achieve the plateau period in loss of weight, 5 mg/day dapagliflozin was required for at least 41.6 weeks.
Assuntos
Compostos Benzidrílicos/uso terapêutico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Glucosídeos/uso terapêutico , Hipoglicemiantes/uso terapêutico , Redução de Peso/efeitos dos fármacos , Fatores Etários , Compostos Benzidrílicos/administração & dosagem , Peso Corporal , Relação Dose-Resposta a Droga , Glucosídeos/administração & dosagem , Humanos , Hipoglicemiantes/administração & dosagem , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos RetrospectivosRESUMO
Gene delivery systems play a vital role in gene therapy and recombinant protein production. The advantages of using gene delivery reagents for non-viral vector include the capacity to accommodate a large packaging load and their low or absent immunogenicity. Furthermore, they are easy to produce at a large scale and preserve. Gene delivery reagents for non-viral vector are commonly used for transfecting a variety of cells and tissues. It is mainly composed of liposomes and non-liposome cationic polymers. According to the different head structures used, the non-viral cationic transfection reagents include a quaternary ammonium salt, amine, amino acid or polypeptide, guanidine salt, and a heterocyclic ring. This article summarizes these approaches and developments of types and components of transfection reagents and optimization of gene delivery. The optimization of mammalian cell transient recombinant protein expression system and cationic reagents for clinical or clinical trials are also discussed.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Animais , Cátions , Indicadores e Reagentes , TransfecçãoRESUMO
Isoliquiritigenin (ILG) and isoliquiritin (ILQ), two kinds of major flavonoids in licorice, are biological active substances with antioxidant, anti-inflammatory, and tumor-suppressive effects. However, their in vivo metabolites, possible material basis of this two licorice chalcones for the treatment of diseases, have not been studied completely. To determine the metabolism of ILG and ILQ, after oral administration of 100 mg/kg/day of these compounds for consecutive 8 days, the metabolites of these two licorice chalcones in mice plasma, urine, feces, and bile were determined using liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry in this study. The structures of those metabolites were tentatively identified according to their fragment pathways, accurate masses, characteristic product ions, metabolism law, and reference standards-matching. As a result, a total of 25 and 29 metabolites of ILG and ILQ were identified, respectively. Seven main metabolic pathways, oxidation and reduction, deglycosylation and glycosylation, dehydroxylation and hydroxylation, demethoxylation and methoxylation, acetylation, glucuronidation, and sulfation, were summarized to tentatively explain how the metabolites were biologically transformed. These results provide the important information on the metabolism of ILG and ILQ, which may be helpful for the further research of their pharmacological mechanism.
Assuntos
Chalcona/análogos & derivados , Chalconas/análise , Cromatografia Líquida/métodos , Glucosídeos/análise , Espectrometria de Massas em Tandem/métodos , Administração Oral , Animais , Bile/química , Chalcona/administração & dosagem , Chalcona/análise , Chalcona/química , Chalcona/farmacocinética , Chalconas/administração & dosagem , Chalconas/química , Chalconas/farmacocinética , Fezes/química , Glucosídeos/administração & dosagem , Glucosídeos/química , Glucosídeos/farmacocinética , Glycyrrhiza , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Mammalian cells are the preferred choice system for the production of complex molecules, such as recombinant therapeutic proteins. Although the technology for increasing the yield of proteins has improved rapidly, the process of selecting, identifying as well as maintaining high-yield cell clones is still troublesome, time-consuming and usually uncertain. Optimization of expression vectors is one of the most effective methods for enhancing protein expression levels. Several commonly used chromatin-modifying elements, including the matrix attachment region, ubiquitous chromatin opening elements, insulators, stabilizing anti-repressor elements can be used to increase the expression level and stability of recombinant proteins. In this review, these chromatin-modifying elements used for the expression vector optimization in mammalian cells are summarized, and future strategies for the utilization of expression cassettes are also discussed.
Assuntos
Cromatina/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Vetores Genéticos/genética , Células HEK293 , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Regiões Promotoras Genéticas , Animais , Células CHO , Cricetinae , Cricetulus , Escherichia coli , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismoRESUMO
Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.
Assuntos
Vetores Genéticos/genética , Peptídeos/genética , Proteínas Recombinantes/genética , Transgenes/genética , Animais , Células CHO , Cricetinae , Cricetulus , Dosagem de Genes , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Human tissue plasminogen activator was the first recombinant therapy protein that successfully produced in Chinese hamster ovary cells in 1986 and approved for clinical use. Since then, more and more therapeutic proteins are being manufactured in mammalian cells, and the technologies for recombinant protein production in this expression system have developed rapidly, with the optimization of both upstream and downstream processes. One of the most promising strategies is expression vector cassette optimization based on the expression vector cassette. In this review paper, these approaches and developments are summarized, and the future strategy on the utilizing of expression cassettes for the production of recombinant therapeutic proteins in mammalian cells is discussed.
Assuntos
Vetores Genéticos/genética , Proteínas Recombinantes/biossíntese , Animais , Linhagem Celular , Epigênese Genética , Expressão Gênica , Engenharia Genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Elementos Reguladores de TranscriçãoRESUMO
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.
Assuntos
Clonagem Molecular/métodos , Proteínas Recombinantes/metabolismo , Transgenes , Animais , Células CHO , Cricetinae , Cricetulus , Expressão Gênica , Regiões de Interação com a Matriz , Regiões Promotoras Genéticas , TransfecçãoRESUMO
Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR-3 and MAR-7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real-time PCR. The results showed that the expression level of eGFP of cells transfected with MAR-containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR-7 was higher than that of MAR-3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR-3 and MAR-7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.
Assuntos
Variações do Número de Cópias de DNA/genética , Genoma Humano/genética , Regiões de Interação com a Matriz/genética , Animais , Células CHO , Cricetulus , Regulação da Expressão Gênica no Desenvolvimento/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Humanos , Transfecção , Transgenes/genéticaRESUMO
Matrix attachment regions (MARs) can enhance transgene expression levels and maintain stability. However, the consensus sequence from MARs and its functional analysis remains to be examined. Here, we assessed a possible consensus sequence from MARs and assessed its activity in stably transfected Chinese hamster ovary (CHO) cells. First, we analyzed the effects of 10 MARs on transfected CHO cells and then analyzed the consensus motifs from these MARs using a bioinformatics method. The consensus sequence was synthesized and cloned upstream or downstream of the eukaryotic vector. The constructs were transfected into CHO cells and the expression levels and stability of enhanced green fluorescent protein were detected by flow cytometry. The results indicated that eight of the ten MARs increased transgene expression in transfected CHO cells. Three consensus motifs were found after bioinformatics analyses. The consensus sequence tandemly enhanced transgene expression when it was inserted into the eukaryotic expression vector; the effect of the addition upstream was stronger than that downstream. Thus, we found a MAR consensus sequence that may regulate the MAR-mediated increase in transgene expression.
Assuntos
Sequência Consenso/genética , Regiões de Interação com a Matriz/genética , Transfecção , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Dosagem de Genes , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Recombinantes/metabolismo , TransgenesRESUMO
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.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Animais , Células CHO , Biologia Computacional , Cricetinae , Cricetulus , Vetores Genéticos/genética , Glutationa/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Estabilidade Proteica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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.
Assuntos
DNA/genética , Terapia Genética , Plasmídeos/genética , Transgenes/genética , Animais , Células CHO , Cricetinae , Cricetulus , Metilases de Modificação do DNA/genética , Vetores Genéticos/genética , Regiões de Interação com a Matriz/genética , Regiões Promotoras Genéticas , TransfecçãoRESUMO
OBJECTIVES: To investigate the effect of full-length fragment of DNA topoisomerase I gene (TOP1) matrix attachment regions (MARs) originating from the human genome on transgene expression in Chinese hamster ovary (CHO) cells and explore the underlying mechanisms. RESULTS: Results showed that TOP1 MAR cannot only enhance the transient and stable transgenic expression of enhanced green fluorescence protein (EGFP) but also increase long-term stability and ratio of positive colonies in transfected CHO cells with TOP1 MAR at the 5' or 3' ends of the EGFP expression cassette. Interestingly, the CHO cells were transfected with the 5',3' TOP1 MAR-containing vector featured the highest transient and stable expression, whereas those with the 3' TOP1 MAR-containing vector exhibited the most effective stability and ratio of positive colonies. We also observed that transgene copy numbers and mRNA of egfp gene were correlated with the expression levels of EGFP protein in polyclonal CHO cells. However, the heterogeneity of expression in monoclonal CHO cells was unaffected by transgene copy number. CONCLUSIONS: The findings may aid in the potential application of TOP1 MAR in expression enhancement of recombinant proteins in mammalian cells.
Assuntos
Células CHO , Engenharia Celular/métodos , DNA Topoisomerases Tipo I/genética , Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Regiões de Interação com a Matriz , Proteínas Recombinantes/biossíntese , Animais , Cricetulus , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Recombinantes/genéticaRESUMO
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.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , DNA (Citosina-5-)-Metiltransferases/genética , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos/genética , Transgenes , Animais , Sequência de Bases , Células CHO , Proteína 9 Associada à CRISPR/metabolismo , Ilhas de CpG , Cricetulus , Citomegalovirus/genética , Citomegalovirus/metabolismo , DNA (Citosina-5-)-Metiltransferases/deficiência , Metilação de DNA , Expressão Gênica , Técnicas de Inativação de Genes , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
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.
Assuntos
Íntrons/genética , Vírus 40 dos Símios/genética , Transfecção/métodos , Transgenes , Animais , Células CHO , Cricetinae , Cricetulus , Eritropoetina/metabolismo , Dosagem de Genes , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Low-level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR-6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR-6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP-B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP-B, DHFR intron MAR element and MAR-6. Additionally, as expected, the three MAR-containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non-MAR-containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP-B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.
Assuntos
Genoma Humano , Regiões de Interação com a Matriz/genética , Transfecção , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Dosagem de Genes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , TransgenesRESUMO
Recent years have seen the use of recombinant proteins in the treatment of different diseases. Among them, monoclonal antibodies (mAbs) are currently the fastest growing class of bio-therapeutic recombinant proteins. Chinese hamster ovary (CHO) cells are the most commonly used host cells for production of these recombinant mAbs. Expression vectors determine the expression level and quality of recombinant mAbs. Currently, few construction strategies for recombinant mAbs expression vectors in CHO cells have been developed, including monocistronic vector, multiple-promoter expression vector, and tricistronic vector mediated by internal ribosome entry site (IRES) or Furin-2A element. Among them, Furin-2A-mediated vector is an effective approach due to advantages of high "self-cleavage" efficiency, and equal expression of light and heavy chains from a single open reading frame. Here, we have reviewed the progress in development of different strategies for constructing recombinant mAb expression vectors in CHO cells and its potential advantages and disadvantages.
Assuntos
Anticorpos Monoclonais/genética , Clonagem Molecular/métodos , Engenharia de Proteínas/métodos , Animais , Anticorpos Monoclonais/fisiologia , Formação de Anticorpos/genética , Células CHO , Cricetulus , Vetores Genéticos/síntese química , Vetores Genéticos/genética , Humanos , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Transfecção/métodosRESUMO
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.