Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells

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.     

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 β‐globin MAR in different sites, including the vector with two MARs flanking the CAT (chloramphenicol acetyltransferase) expression cassette, one MAR at the 5′ or 3′ 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 5′ site upstream of the expression cassette, whereas the transgene expression level decreased when MAR was inserted at the 3′ 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.     

Identification of consensus sequence from matrix attachment regions and functional analysis of its activity in stably transfected Chinese hamster ovary cells

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.     

Enhanced expression of transgene in CHO cells using matrix attachment region

The expression of transgenes in mammalian cells is often at a low level mainly due to position effects from the neighboring chromatin context. To improve this, we have constructed a vector pCAM, which contains chloramphenicol acetyltransferase (CAT) reporter gene cassettes, driven by SV40 early promoter and flanked by two human beta-globin MARs in cis. We transfected this vector into the Chinese hamster ovary (CHO) cell line, and found that the level of CAT gene expression with MAR was effectively increased, about 5.493-fold higher than those without MARs. Moreover, the variations of CAT expression among individuals of transformants were decreased 2.670-fold. Our result also showed that MAR could increase the proportion of positive colonies in recombinants.     

SV40 intron,a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

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.     

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells

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.     

Matrix attachment regions (MARs) enhance transformation frequency and transgene expression in poplar

We tested the value of a matrix attachment region (MAR) fragment derived from a tobacco gene for increasing the frequency of Agrobacterium-mediated transformation. A binary vector that carried a GUS reporter gene containing an intron and an nptII gene was modified to contain flanking MAR elements within the T-DNA borders. Vectors containing or lacking MARs were then used to transform tobacco, a readily transformabl e poplar clone (Populus tremula × P. alba), and a recalcitrant poplar clone (Populus trichocarpa × P. deltoides). MARs increased GUS gene expression approximately 10-fold in the two hybrid poplar clones and twofold in tobacco one month after cocultivation with Agrobacterium; MARs also increased the frequency of kanamycin-resistant poplar shoots recovered     

核基质结合序列（MAR）与基因表达调控

核基质结合序列（MAR）是能在体外与核基质特异结合的DNA序列,广泛存在于染色质Loop结构的边界序列中。随着研究的深入,发现MAR序列不仅在染色质折叠中起到重要作用,影响邻近内源基因表达,而且将MAR序列构建到外源基因表达盒两侧转化动植物时,也影响外源基因的表达。因此,MAR序列是基因组中一种重要的基因间边界序列,为阐明非编码序列在基因表达中的作用和构建真核生物高效表达载体提供了新途径。     

A short synthetic chimeric sequence harboring matrix attachment region/PSAR2 increases transgene expression in Chinese hamster ovary cells

A chimeric DNA fragment containing an interferon-beta matrix attachment region (MAR) and an immunoglobulin MAR (PSAR2) was synthesized. PSAR2 was cloned into the upstream or downstream region of an enhanced green fluorescent protein (eGFP) expression cassette in a eukaryotic vector, which was then transfected into CHO cells. The results showed that PSAR2 did not effectively increase transgene expression when it was cloned into the upstream region of the eGFP expression cassette. However, when inserted downstream of the eGFP expression cassette, PSAR2-enhanced transient transgene expression and significantly increased the numbers of stably transfected cells compared with the control vector. Additionally, PSAR2 significantly increased eGFP copy numbers as compared with the control vector. PSAR2 could significantly enhance transgene expression in CHO cells according to the position in the vector and increased transgene copy numbers. We found a short chimeric sequence harboring two MARs effectively increased transgene expression in CHO cells.     

植物的MAR及其对转基因表达的效应

与核基质结合的DNA序列称为基质附着区（matrix attachment regions,MARs）,可提高转基因的表达水平并降低转基因在不同转基因系间的表达差异,因其在植物基因工程中的巨大应用潜力而引起了研究者的极大兴趣。对植物中MAR的研究尚处于早期阶段,本文综述了植物中MAR的分离鉴定、序列特征及MAR对植物中转基因表达的影响,并进一步讨论了MAR对转基因效应的可能机制。 Abstract：DNA sequences called matrix attachment regions (MARs) have recently attracted mu ch attention because of their perceived capacity to increase levels of transgene expression and to reduce transformant-to-transformant variation of transgene ex pression in plants. Work with MARs in plants is in its early stage .In the prese nt paper ,we reviewed the procedure to isolate and identify MAR sequences from higher plants, the sequence characteristics of the plant MARs and the effect of MARs on the transgene expression in plants. Funthermore, the possible mechanism to explain how MARs affect transgene expression in transformants was discussed.     

玉米核基质结合区在烟草中对外源转基因表达水平的影响

为研究核基质结合区(matrix attachment region, MAR)在转基因植物中的功能,将来自玉米基因组的MAR序列构建在植物表达载体T-DNA中, 并将报告基因β-葡糖醛酸酶(β-glucuronidase, GUS)基因(uidA)插入两段MARs序列之间.将此载体与不包含MARs序列的植物表达载体分别转化烟草(Nicotiana tabacum L.).GUS活性检测表明,MARs可以显著提高外源基因uidA在转基因烟草中的表达水平,平均表达水平提高2倍,最高单株活性可达10倍.并且转基因植株GUS活性高低与稳定mRNA的量成正比,表明MARs在转录水平提高基因表达.     

CRISPR/Cas9‐mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long‐term stability

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.     

Novel short synthetic matrix attachment region for enhancing transgenic expression in recombinant Chinese hamster ovary cells

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.     

MAR提高重组CHO细胞中转基因表达的分子特征分析

分析核基质结合区（matrix attachment region,MAR）调控转基因表达的分子序列特征,鉴定能有效提高CHO细胞转基因表达的MAR特征性元件。将人β-珠蛋白MAR片段从5′到3′ 端分为6个分段（1～540,421～1 020,901～1 500,1 381～1 980,1 861～2 460,2 341～2 999位置）,分别采用PCR进行克隆,经测序证实正确后,分别连接到含有氯霉素乙酰转移酶（chloramphenicol acetyltransferase,CAT）报告基因的表达载体SV40启动子及上游,构建β-珠蛋白MAR渐次片段介导的表达载体,转染CHO细胞,G418筛选出稳定表达细胞株,ELISA分析CAT报告基因的表达水平,生物信息学分析MAR序列特征。结果表明,β-珠蛋白MAR全长能显著提高转基因的表达,6个渐次片段相比较,421～1 020位的第2个分段和 901～1 500位的第3个分段提高转基因表达作用显著。生物信息学分析结果显示,MAR-like motif有助于转基因表达提高。     

Elevation of Transgene Expression Level by Flanking Matrix Attachment Regions (MAR) is Promoter Dependent: A Study of the Interactions of Six Promoters with the RB7 3′ MAR

We have analyzed effects of a matrix attachment region (MAR) from the tobacco RB7 gene on transgene expression from six different promoters in stably transformed tobacco cell cultures. The presence of MARs flanking the transgene increased expression of constructs based on the constitutive CaMV 35S, NOS, and OCS promoters. Expression from an induced heat shock promoter was also increased and MARs did not cause expression in the absence of heat shock. There was also no effect of MARs on the pea ferredoxin promoter, which is not normally expressed in this cell line. Importantly, most transgenes flanked by RB7 MAR elements showed a large reduction in the number of low expressing GUS transformants relative to control constructs without MARs.     

核基质结合区对转基因表达的影响及其作用机制

核基质结合区(matrix attachment region,MAR)是一段在体外能与核基质结合的富含AT的DNA序列。研究发现MAR能使染色质形成环状结构;将其连到目的基因二侧构建载体并转至生物体中,发现它能增强基因转录表达水平及稳定性,在一定程度上降低转基因个体或细胞系之间转基因的表达水平的差异,这很可能是减低了基因沉默所致。现对MAR的序列特征、MAR对转基因表达的影响及对转基因效应的影响机制进行综述。     

Transient gene expression in CHO cells monitored with automated flow cytometry

Transient gene expression is frequently used in industry to rapidly generate usable quantities of a protein from cultured cells. In gene therapy applications it is used to express a therapeutic protein in vivo. A quantitative assessment of the expression kinetics is important because it enables optimization and control of culture conditions for higher productivity. Previous experimental studies show a characteristic peak in average protein expression per cell after transfection followed by an exponential decrease of the expressed protein. Here, we show that the exponential decrease in single cell expression of enhanced Green Fluorescent Protein (eGfp) occurs in discrete steps. We attribute this to the absence of plasmid replication and to symmetric partitioning of plasmid and eGfp between dividing cells. This is reflected in the total eGfp in the bioreactor, which increased at a constant rate throughout the experiment. Additionally, the data provide a detailed time course of cell physiology during recovery from electroporation. The time course of cell physiology precisely indicates when the culture shifts growth phases. Furthermore, the data indicate two unique stationary phases. One type of stationary phase occurs when proliferation ceases while cells decrease their cell size, maintain granularity, and mean eGfp content decreases. The second type occurs when proliferation ceases while cells increase their cell size, increase granularity, and surprisingly maintain eGfp content. The collected data demonstrate the utility of automated flow cytometry for unique bioreactor monitoring and control capabilities in accordance with the US Food and Drug Administration’s Process Analytical Technology initiative.