How a Replication Origin and Matrix Attachment Region Accelerate Gene Amplification under Replication Stress in Mammalian Cells

The gene amplification plays a critical role in the malignant transformation of mammalian cells. The most widespread method for amplifying a target gene in cell culture is the use of methotrexate (Mtx) treatment to amplify dihydrofolate reductase (Dhfr). Whereas, we found that a plasmid bearing both a mammalian origin of replication (initiation region; IR) and a matrix attachment region (MAR) was spontaneously amplified in mammalian cells. In this study, we attempted to uncover the underlying mechanism by which the IR/MAR sequence might accelerate Mtx induced Dhfr amplification. The plasmid containing the IR/MAR was extrachromosomally amplified, and then integrated at multiple chromosomal locations within individual cells, increasing the likelihood that the plasmid might be inserted into a chromosomal environment that permits high expression and further amplification. Efficient amplification of this plasmid alleviated the genotoxicity of Mtx. Clone-based cytogenetic and sequence analysis revealed that the plasmid was amplified in a chromosomal context by breakage-fusion-bridge cycles operating either at the plasmid repeat or at the flanking fragile site activated by Mtx. This mechanism explains how a circular molecule bearing IR/MAR sequences of chromosomal origin might be amplified under replication stress, and also provides insight into gene amplification in human cancer.     

Efficient Recombinant Production in Mammalian Cells Using a Novel IR/MAR Gene Amplification Method

We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone.     

Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.     

All-<Emphasis Type="Italic">trans</Emphasis>- and 9-<Emphasis Type="Italic">cis</Emphasis>-retinoic acids activate the human cyclooxynase-2 gene: a role for DR1 as RARE or RXRE

The human COX-2 promoter contains a direct repeat 1 (DR1) which was shown to confer responsiveness to PPARs. We found that in AN₃CA and F9 cells, this hCOX-2 DR1 mediates responsiveness to all-trans-retinoic acid (tRA) or 9-cis-retinoic acid (9cRA), but this effect was suppressed by PPARδ. Truncated PPARδ lacking the activation domain AF2 cannot suppress RA-induced activation of the hCOX-2 gene via DR1, suggesting that cofactor recruitment by AF2 is required for the suppression by PPARδ. Gel shift assay showed that PPAR/RXR, RARβ/RXR, and RXR/RXR, bind to hCOX-2 DR1, revealing the promiscuity of this DR1. Particularly, RXR homodimer was able to bind to this DR1 only in the presence of 9cRA. Our results established that tRA and 9cRA are potent inducers of hCOX-2 and that the hCOX-2 DR1 could either serve as RARE or RXRE depending on cellular contexts.     

Dissection of the Beta-Globin Replication-Initiation Region Reveals Specific Requirements for Replicator Elements during Gene Amplification

Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR)/origin/replicator and a nuclear matrix-attachment region (MAR) is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR). Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.     

一种1型单纯庖疹病毒载体系统的建立

1型单纯疱疹病毒（HSV-1）作为溶瘤病毒和病毒载体的研究已有很长的历史. 本研究利用细菌人工染色体技术建立了一种HSV-1载体系统. 首先,将HSV-1内部反向重复序列（internal inverted repeat sequences, IR）两侧的片段克隆入pKO5获得穿梭质粒pKO5/BN,其电转含pHSV-BAC的大肠杆菌后筛选获得删除IR区重组DNA的 pHSVΔIR-BAC. pHSVΔIR-BAC转染Vero细胞获得删除IR区的重组病毒HSVΔIR（MH1001）.上述pKO5/BN和含pHSVΔIR BAC的大肠杆菌构成了HSV-1载体系统. 利用该系统获得了表达绿色荧光蛋白EGFP的重组病毒HSVΔIR/EGFP（MH1002）.MH1001和MH1002在感染的Vero细胞中增殖水平略低于野生型HSV-1,但无显著差异|Western印迹检测表明,重组病毒早期蛋白质ICP0、ICP4、ICP8、ICP22、ICP27在感染细胞中的表达水平下降|免疫荧光及激光共聚焦检测表明,重组病毒与野生型病毒均存在于细胞质中.以上结果表明,删除IR区的重组HSV-1保留了复制能力,能够携载并表达外源基因,建立的HSV-1载体系统可用于构建携载外源基因的复制型重组HSV-1.     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因（k2tPA）、扩增基因（dhfr）、重组酶识别序列（FRT）及筛选基因（neo）,且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO／dhfr^-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点（Hotspot）区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO／dhfr-细胞系。随后利用位点特异性重组系统（FLP-FRT）将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17．1μg／10^6cell·24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

Cyclooxygenase (COX)-2 and COX-1 potentiate beta-amyloid peptide generation through mechanisms that involve gamma-secretase activity

In previous studies we found that overexpression of the inducible form of cyclooxygenase, COX-2, in the brain exacerbated beta-amyloid (A beta) neuropathology in a transgenic mouse model of Alzheimer's disease. To explore the mechanism through which COX may influence A beta amyloidosis, we used an adenoviral gene transfer system to study the effects of human (h)COX-1 and hCOX-2 isoform expression on A beta peptide generation. We found that expression of hCOXs in human amyloid precursor protein (APP)-overexpressing (Chinese hamster ovary (CHO)-APPswe) cells or human neuroglioma (H4-APP751) cells resulting in 10-25 nM prostaglandin (PG)-E2 concentration in the conditioned medium coincided with an approximately 1.8-fold elevation of A beta-(1-40) and A beta-(1-42) peptide generation and an approximately 1.8-fold induction of the C-terminal fragment (CTF)-gamma cleavage product of the APP, an index of gamma-secretase activity. Treatment of APP-overexpressing cells with the non-selective COX inhibitor ibuprofen (1 microM, 48 h) or with the specific gamma-secretase inhibitor L-685,458 significantly attenuated hCOX-1- and hCOX-2-mediated induction of A beta peptide generation and CTF-gamma cleavage product formation. Based on this evidence, we next tested the hypothesis that COX expression might promote A beta peptide generation via a PG-E2-mediated mechanism. We found that exposure of CHO-APPswe or human embryonic kidney (HEK-APPswe) cells to PG-E2 (11-deoxy-PG-E2) at a concentration (10 nM) within the range of PG-E2 found in hCOX-expressing cells similarly promoted (approximately 1.8-fold) the generation of the CTF-gamma cleavage product of APP and commensurate A beta-(1-40) and A beta-(1-42) peptide elevation. The study suggests that expression of COXs may influence A beta peptide generation through mechanisms that involve PG-E2-mediated potentiation of gamma-secretase activity, further supporting a role for COX-2 and COX-1 in Alzheimer's disease neuropathology.     

Improved recombinant gene expression in CHO cells using matrix attachment regions

The use of animal cells such as Chinese hamster ovary (CHO) cells for recombinant gene expression provides many advantageous features such as proper folding and post-translational modification of the recombinant protein. However, recombinant genes introduced into animal cells are often expressed at low levels mainly due to position effects from the neighboring chromatin context. The tedious and time-consuming selection and amplification procedure has been the major hurdle for using animal cell line such as CHO cells. To improve mammalian cell expression systems, we screened a variety of matrix/scaffold attachment region (MAR/SAR) elements for their ability to insulate transgene expression from the position effects in CHO cells. We found that the human beta-globin MAR element is particularly effective as the frequency of beta-Gal positive colonies was increased by up to 80%. The expression levels of these colonies were also enhanced about seven-fold. These improvements appear to be related to the increased copy numbers and a higher efficiency of expression of the integrated genes. When this element was used to express soluble TGF-beta type II receptor (sTbetaRII) through the gene amplification system, the frequency of colonies expressing detectable amounts of sTbetaRII was much higher than that of the control vector. We could also generate high sTbetaRII producers with uniform growth properties by a simple two-step amplification process involving two concentrations of methotrexate. This eliminates the need to isolate individual colonies followed by multi-step treatments of methotrexate and thereby greatly simplifies this mammalian expression system.     

中国老年心脑血管疾病患者阿司匹林抵抗与环氧化酶-1单体型关联分析

目的:旨在研究老年心脑血管病患者阿司匹林抵抗与阿司匹林作用靶点环氧化酶-1(COX-1)基因单体型的关联性。方法:入选北京地区服用阿司匹林的老年汉族心脑血管疾病患者431例,通过花生四烯酸诱导光比浊法,筛选出阿司匹林抵抗患者59例作为病例组,372例阿司匹林不抵抗者作为对照组,使用美国Sequenom系统SNP分型技术鉴定了COX-1基因6个常见单核苷酸多态性位点(SNP)与阿司匹林抵抗的关联性。6个SNP分别是:rs1888943(8759C/T)、rs1330344(1676A/G)、rs3842787(exon 2,50C/T,p.Pro17Leu)、rs5787(exon 4,323G/A,p.Arg108Gln)、rs5789(exon7,709C/A,p.Leu237Met)和rs5794(exon10,1330G/A,p.Val481Ile)。结果:突变COX-1单体型CGCGCC,在病例组频率为0.48(57/118),对照组频率为0.39(286/742),显著高于对照组(P≤0.05)。结论:中国老年汉族心脑血管疾病患者阿司匹林抵抗与COX-1单体型相关联,突变COX-1单体型CGCGCC显著增加了阿司匹林抵抗的发病风险。     

Expression of recombinant cyclooxygenase 1 in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> S2 cells transformed with human β1,4-galactosyltransferase and Galβ1,4-GlcNAc α2,6-sialyltransferase

We examined the expression of human cyclooxygenase-1 (COX-1) in Drososphila melanogaster S2 (S2) cells transformed with cDNAs encoding β1,4-galactosyltransferase (GalT) and Galβ1,4-GlcNAc α2,6-sialyltransferase (ST). Southern blot analysis indicated that multiple copies of the glycosyltransferases genes were integrated into the S2 cell genome. A lectin blot analysis also indicated that recombinant COX-1 from S2COX-1/GalT-ST cells contained the glycan residues of β1,4-linked galactose and α2,6-linked sialic acid. The specific peroxidase activity of recombinant sialylated COX-1 from S2COX-1/GalT-ST cells was 41,250 U mg⁻¹, indicating an increase of approximately 22% compared with a non-sialylated control (33,850 U mg⁻¹) from S2COX-1 cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electric field mediated transformation: isolation and characterization of a TK+ subclone

Transformation of mammalian TK- cells by a plasmid carrying the TK gene from Herpes virus simplex 1 (pAGO) was mediated by electroporation. The cells were treated either in suspension or growing in monolayers directly in the petri dish. The yield of transformation was between 8.10(-5) and 2.10(-4) per microgram DNA depending on the experimental conditions. The structure of the integrated DNA was investigated proving the occurrence of a duplication process that affected preferentially the pBR322 part of the pAGO DNA (60 copies per cell). The TK gene that gave the TK+ phenotype to the selected clone was present in less than 6 copies.     

HPV16l1基因的克隆及其在真核细胞中的表达

克隆并表达人乳头瘤病毒16型(HPV16)晚期基因l1,以期为研制防治宫颈癌的DNA疫苗奠定基础。本实验采用PCR方法从质粒p16L1BN1中获得HPV16l1基因片段,利用基因重组技术,将其克隆至含巨细胞病毒(CMV)启动子的真核表达载体中,核酸序列鉴定HPV16l1基因真核表达质粒构建成功,再用脂质体介导基因转染7721人肝癌细胞。转化阳性细胞经SDS-PAGE显示在分子量大约为55kDa的位置出现一条特异性条带,与HPV16L1分子量大小相符。表达产物经Western blotting分析:能与HPV16L1单克隆抗体特异结合。真核表达质粒pcDNA3-HPV16L1构建成功并能在真核细胞7721中有效表达,为下一步进行动物DNA免疫实验奠定了基础。     

Sphingosine-1-phosphate induces COX-2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX-2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX-2 expression by S1P in VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced the expression of COX-2 mRNA and protein in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen-activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX-2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by a selective NF-kappaB inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappaB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P-stimulated activation of NF-kappaB promoter activity was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF-kappaB. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappaB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappaB pathways was essential for S1P-induced COX-2 gene expression. Understanding the mechanisms involved in S1P-induced COX-2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.     

Molecular cloning, expression and characterization of the Canis familiaris interleukin-4.

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. The rapid amplification of cDNA ends (RACE) was used to clone the canine IL-4 gene. It was expressed in mammalian cells and Escherichia coli. Monoclonal antibodies were raised to rcIL-4 for use in an enzyme-linked immunosorbent assay (ELISA). This will facilitate studies on the role of cIL-4 in inflammatory diseases, particularly rheumatoid arthritis.