Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells.

Overlapping recombinant lambda 1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been isolated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly repeated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolutionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.     

Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells

We constructed a gene library from a murine cell line with amplified dihydrofolate reductase (dhfr) genes by inserting random segments of DNA into lambda Ch4A. From this library, the dhfr gene and 30 kilobase pairs of surrounding DNA were cloned, and the restriction map was determined. All of the coding regions were sequenced and show that the gene spans a total of 31 kilobase pairs and has five intervening sequences in the coding portion of the gene. In addition, two classes of variant dhfr genes were found in the amplified line, which were amplified and present at levels of 10 to 30% of the normal dhfr genes. Numerous repeated sequences were located throughout the gene region, some of which share homology with previously defied families of repeats.     

Pmscv-Ubc9逆转录病毒表达载体的构建及产毒细胞系的建立

目的：构建含Ubc9的逆转录病毒表达载体,筛选建立携带该基因的高滴度产毒细胞系,深入研究SUMO化修饰的作用。方法：聚合酶链反应（PCR）扩增获取目的基因Ubc9,定向插入逆转录病毒表达载体pMSCVneo,形成重组质粒pMSCV-Ubc9;脂质体法将pMSCV-Ubc9转染逆转录病毒包装细胞PT67;G418筛选产毒细胞克隆,扩大培养产毒细胞克隆,收获病毒感染NIH3T3细胞。结果：限制性酶切和测序鉴定证实Ubc9正确插入逆转录病毒表达载体。G418筛选获得稳定产毒的抗性细胞克隆,收获病毒能有效感染NIH3T3细胞。结论：携带Ubc9基因的重组逆转录病毒表达载体pMSCV-Ubc9构建成功,转染PT67细胞后包装出重组逆转录病毒,进而筛选获得了能转录表达Ubc9的产毒细胞系PT67-Ubc9。     

Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line

Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins.     

Transformation of the cells of transplantable teratocarcinoma CBA9H6 by a bacterial dihydrofolate reductase gene

A plasmid containing the bacterial gene of methotrexate-resistant dihydrofolate reductase (dhfr), under the control of early SV40 promoter, was introduced into murine teratocarcinoma CBA9H6 cells. From the whole pool of teratocarcinoma cells, which survived after transient methotrexate selection in vivo, the individual cells were isolated to give rise to 15 clones of tumors. Six of the 15 clones displayed nucleotide sequences of the original vector containing pBR322 sequences and the early SV40 promoter region; however, the bacterial dhfr gene was absent from the transformant clones. Possible causes of the loss of introduced dhfr gene from teratocarcinoma cells under non-selective conditions are discussed.     

A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein

A new series of double-selection plasmids containing recombinant genes expressing the neomycin phosphotransferase (NEO) of transposon Tn5 and mouse dihydrofolate reductase (DHFR) in mammalian cells is described. Activity of the recombinant DHFR gene varied more than 50-fold, depending on the location of the simian virus 40 72 base-pair repeat or enhancer, which is part of the promoter of the NEO unit. A NEO-DHFR module with the enhancer located at the 3' end of the DHFR gene was inserted into a plasmid containing four tandem head-to-tail copies of the hepatitis B virus (HBV) genome and the new plasmid was used to transform DHFR- Chinese hamster ovary cells. In one of the cell lines obtained, an unrearranged copy of the HBV tetramer could be amplified 300-fold by increasing selective pressure with methotrexate, resulting in a proportional increase of the synthesis of HBV surface antigen. Four different mRNAs detected in the amplified cell line probably encode HBV core protein, pre-S and surface antigens, and the X protein. As a result of the DNA amplification, synthesis of HBV proteins is no longer restricted to resting cells. Integrated plasmid sequences appear to be stable during the amplification process.     

A genetic test for expression of a functional herpes simplex virus DNA-binding protein from a transfected plasmid.

The major DNA-binding protein, ICP8, encoded by herpes simplex virus is localized to the infected cell nucleus where it plays a role in viral DNA replication and control of viral gene expression. To identify the parts of the ICP8 protein that are important for its localization and functions, we have developed a system to test the ability of recombinant plasmids to express functional ICP8. A recombinant plasmid containing the wild-type ICP8 gene was transfected into cells. The cells were later infected with a temperature-sensitive ICP8 mutant virus at the nonpermissive temperature. Sufficient wild-type ICP8 was expressed from the transfected plasmid to complement the replication of the mutant virus. This provides a genetic system to test the properties of ICP8 expressed from mutagenized plasmids without the establishment of a stable cell line or the reintroduction of the ICP8 gene into the herpes simplex virus genome.     

Generation of recombinant CHO(dhfr-) cell lines by single selection for dhfr+ transformants.

In order to establish a mammalian cell expression system with a minimum of selection steps and a stable expression of microgram amounts of recombinant protein (human tissue-type plasminogen activator mutants and chimeric proteins) per 10(6) cells per day, we investigated Chinese hamster ovary cells and the dihydrofolate reductase-deficient Chinese hamster ovary cell line CHO(dhfr-). The 1tPA expression vector pCMVtPA was cotransfected either with the SV40 enhancer sequence containing dhfr expression vector pMT2 or with the enhancerless dhfr expression vector pAdD26SV(A) into CHO(dhfr-) cells. With both dhfr expression plasmids, selection for dhfr+ transformants followed by single dilution cloning was sufficient to generate cell lines with a production level of up to 4.6 micrograms tPA/10(6) cells.day. This approach is useful if gene amplification procedures are time-consuming and impracticable because of a large number of recombinant proteins. In order to establish CHO cell lines with a tPA expression level as high as that in the case of CHO(dhfr-) cells, repeated dilution cloning is necessary.     

Pmscv-Ubc9 逆转录病毒表达载体的构建及产毒细胞系的建立

目的:构建含Ubc9的逆转录病毒表达载体,筛选建立携带该基因的高滴度产毒细胞系,深入研究SUMO化修饰的作用。方法:聚合酶链反应(PCR)扩增获取目的基因Ubc9,定向插入逆转录病毒表达载体pMSCVneo,形成重组质粒pMSCV-Ubc9;脂质体法将pMSCV-Ubc9转染逆转录病毒包装细胞PT67;G418筛选产毒细胞克隆,扩大培养产毒细胞克隆,收获病毒感染NIH3T3细胞。结果:限制性酶切和测序鉴定证实Ubc9正确插入逆转录病毒表达载体。G418筛选获得稳定产毒的抗性细胞克隆,收获病毒能有效感染NIH3T3细胞。结论:携带Ubc9基因的重组逆转录病毒表达载体pMSCV-Ubc9构建成功,转染PT67细胞后包装出重组逆转录病毒,进而筛选获得了能转录表达Ubc9的产毒细胞系PT67-Ubc9。     

Amplification of plasmid (L factor) DNA and increased production of a plasmid gene product (gamma-interferon) in mouse L cells.

It has previously been reported that composite DNAs derived from L factor, a polyoma-related mammalian plasmid, can be established in several mouse cell lines after transfection. Here, we report that the copy number of a plasmid composite DNA consisting of L factor, pBR DNA, dihydrofolate reductase (dhfr) gene, and gamma-interferon (gamma-IFN) gene was increased more than 10-fold after two successive adaptations of the plasmid-bearing mouse L cells to increasing concentrations of methotrexate (MTX), an inhibitor of dhfr. The structure of the amplified L factor plasmid remained intact during prolonged cell culture, but the copy number remained to be amplified only when the selective pressure (presence of MTX in the medium) has been exerted during the culture. Cells bearing the amplified plasmid produced a higher level of gamma-IFN compared with the original clone, which was likely to be derived from the plasmid gamma-IFN gene amplified along with L factor and the dhfr gene.     

Gene Therapy of Lung Adenocarcinoma using Herpes Virus Expressing a Fusogenic Membrane Glycoprotein

The aim of this study is to observe the in vitro-targeted destruction of lung adenocarcinoma using recombinant Type I herpes simplex virus (HSV-I)-mediated gibbon ape leukemia virus envelope glycoprotein (GALV.fus), controlled by UL38 promoter and cytomegalovirus promoter (CMVP). A recombinant HSV-I plasmid encoding the GALV.fus was transfected into green monkey kidney cells, the lung adenocarcinoma line A549, and the human fetal fibroblast cell line HFL-I GNHu5 in various doses. The effects and expression of in vitro GALV.fus were observed using an inverted microscope. Enhanced green fluorescence protein expression served as the contro1 for GALV.fus. Recombinant HSV-I virus was produced. Fusogenic recombinant virus infection led to cell fusions in A549 in a dose-dependent manner. Nonfusogenic viruses only produced conventional cytotoxic effects. Recombinant HSV-I with the CMVP initiated cell fusions in HFL-1 GNHu5 cells with arrested cell cycles or as quiescence. HSV-I regulated by UL38p caused cell fusion only in growing cells. Protein expression of GALV.fus was confirmed by Western Blot in infected A549 and HFL-1 GNHu5. Delivery and tumor-specific expression of GALV.fus gene can selectively and safely target lung cancer in vitro, and may prove to be a novel gene therapy for lung cancer.     

Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.     

TT病毒ORF2在Cos7细胞中的表达及蛋白质定位

应用PCR方法从含有1TrVirusORF2的质粒pET-His-TTV2中扩增出606bp的蛋白质编码区,并将其克隆到真核表达载体pEGFP．NI中以表达成GFP—VP2融合蛋白。构建出的重组质粒pEGFPTFV2经过酶切分析和PCR鉴定。用脂质体介导法将pEGFPTTV2质粒DNA转染Cos7细胞,通过RT-PCR分析,证实细胞中存在ORF2基因的转录产物。用共聚焦显微镜结合PI染色技术研究1TTV P2蛋白在细胞中的分布情况。结果表明,1TrVVP2分布在细胞质中和细胞核膜内侧。因此推测VP2作为一种非结构蛋白,功能可能是参与病毒DNA的复制或转录。     

Mo MLV gag-pol基因在NIH3T3细胞中的表达和鉴定

目的 构建含MoMLV gag-pol基因的重组表达载体,实现其在NIH3T3细胞中稳定表达。方法 应用RT-PCR方法反转录并扩增gag-pol基因,克隆入真核表达载体pcDNA4/HisMaxA上,构建重组表达载体pcDNA4/HisMaxA-gag-pol,用脂质体法转染NIH3T3细胞,Zeocin筛选稳定表达细胞株,通过SDS-PAGE分析检测表明, gag-pol基因在NIH3T3细胞实现了表达,产物相对分子质量(kD)为194.78×103。然后,将逆转录病毒载体导入此细胞系,包装逆转录病毒。用PCR与标记基因补救分析法检测野生型辅助病毒。结果 酶切鉴定的片段大小分别为5.2-kb,与预期大小一致,经Zeocin筛选后获得稳定表达细胞株,SDS-PAGE实验表明产物融合蛋白相对分子质量(kD)为194.78×103,与预期相符。脂质体转染包装细胞,嘌呤霉素加压筛选出高病毒滴度(4.0×106CFU/ml)的细胞克隆,且未检测到辅助病毒。结论 本工作构建的融合表达载体pcDNA4/HisMaxA-gag-pol及其在NIH3T3细胞中的表达,构建成功具有靶向性的逆转录病毒包装细胞系,该细胞系能够包装出高滴度的逆转录病毒,为肝细胞的基因治疗提供了一种新的基因转移系统。     

A new runaway type episomal vector for mammalian cells based on a temperature-sensitive simian virus 40 and inducible erythropoietin production

A runaway vector for mammalian cells was constructed from the simian virus 40 (SV40) genome with a temperature-sensitive mutation of the large T antigen and bacterial neo ^r gene. Replication of this plasmid was repressed above 39°C and vigorous DNA propagation was observed below 33°C in simian CV-1 cells. The human erythropoietin gene was inserted downstream of the SV40 late promoter of the plasmid and the recombinant plasmid was introduced into CV-1 cells. By a temperature shift from 37 to 33°C, the plasmid copy number increased from 5 × 10² to 5 × 10³ copies per cell and the specific production rate of erythropoietin increased more than ten-fold. The bacterial-derived sequences such as the neo ^r gene and vector pUC sequences were prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained at either 33 or 37°C.     

Synthesis and processing of human immunodeficiency virus type 1 envelope proteins encoded by a recombinant human adenovirus.

A recombinant adenovirus was constructed by inserting the human immunodeficiency virus type 1 (HIV-1) envelope gene downstream from the early region 3 (E3) promoter of adenovirus type 5 (Ad5), replacing the coding sequences of E3. The recombinant virus replicated as efficiently as the parent virus in all cell lines tested. Human cells infected with the recombinant virus synthesized the HIV-1 envelope precursor gp160, which was efficiently processed to the envelope glycoproteins gp120 and gp41. A human T-lymphoblast line (Molt-4) infected with the recombinant virus expressed HIV-1 envelope glycoproteins on the cell surface, leading to syncytium formation. The envelope gene was expressed from the E3 promoter at early times after infection and at late times from the major late promoter. When cotton rats were infected with the recombinant virus, antibodies against the HIV-1 envelope glycoproteins could be expressed in an immunoreactive form by the recombinant adenovirus, further illustrating the usefulness of adenoviruses as expression vectors.