SV40载体在COS7细胞中的复制及外源基因的表达

为评价SV4 0载体 COS7稳定表达系统的可能应用价值 ,将含人组织型纤溶酶原激活剂 (t PA)编码区的SV4 0载体pctPA转染COS7细胞 ,并以G4 18选择培养 2 8d ,得到稳定的抗性细胞库 .Southern印迹和溶圈法分别分析该细胞库在随后的细胞传代中细胞内附加体拷贝数及t PA表达水平的变化 .在经选择培养 2 8d的COS7细胞库中 ,质粒pctPA以染色体外附加体的形式存在 (30 0拷贝 细胞 ) ,t PA的表达水平为 1 1μg d(10 6细胞 ) .在随后 3个月的动态观察中 ,随着细胞传代 ,该COS7细胞库tPA的表达水平虽逐渐递减 ,但在第 1个月内可保持在 1μg d(10 6细胞 )以上 .基于SV4 0载体 COS7稳定表达系统无需筛选克隆 ,在稳定的抗性细胞库形成后的 1个月内可能保持目的基因的高水平表达 ,因而较适合于需同时制备多种重组蛋白的实验 .     

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.     

SV 40-transformed normal and DNA-repair-deficient human fibroblasts can be transfected with high frequency but retain only limited amounts of integrated DNA

The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0-40 kb in length. The transfection frequency for the selected (G418R) marker was between 2 x 10(-4) and 2 x 10(-3) for all cell lines, comparable to many other mammalian systems. About 50% of the G418R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.     

Establishment of a temperature-inducible cell line for human plasminogen activator (tissue-type) by transfection of monkey cells with expression constructs

An expression construct for human tissue-type plasminogen activator (t-pA) cDNA [containing a simian virus 40 (SV40) origin of replication] was introduced into CV1, COS-7 and COSts2 cells; in the latter cell line the amount of functionally active large T antigen of SV40 is regulated by the temperature. In a transient system, the expression in COSts2 cells at the permissive temperature for large T antigen was improved sixfold compared to COS-7 cells. By cotransfection with a plasmid conferring resistance to G418 into COSts2 cells, a cell line (COSts2Glob t-pA) could be isolated with barely detectable expression of t-pA at the semi-permissive and non-permissive temperature and inducible secretion of t-pA at the permissive temperature. The kinetics of induction, inducibility after continued propagation at the semi-permissive temperature and the influence of the temperature during previous propagation on inducibility were investigated. The biological activity of the secreted material was demonstrated by a functional assay. Inducibility of t-pA by temperature was accompanied by a dramatic increase of the copy number of episomal plasmids (up to 2000 copies per cell).     

Association of viral and plasmid DNA with the nuclear matrix during productive infection

The association of simian virus 40 (SV40) DNA or plasmid DNA in subcellular fractions from either infected or transfected cells was examined. In lytically infected cells, approx. 25% of viral specific DNA during the infection cycle was retained in nuclei after washing with low ionic strength buffer and 1% Triton X-100. Viral replicating DNA found in the nuclear matrix was capable of performing limited DNA synthesis by the endogenous DNA polymerase in vitro. Viral DNA synthesized in vitro hybridized preferentially to SV40 Hind-III B and C fragments which are in proximity to the origin of replication. In plasmid-transfected COS-7 cells (SV40-transformed cells), the amount of plasmid DNA found in the nuclear matrix was related to its replication efficiency in cells. More than 80% of the plasmid DNA was tightly associated with subnuclear structures. Little or no plasmid DNA was found in the cytoplasmic fraction. The results suggest that, in extrachromosomal model systems, the association of DNA with nuclear matrix is important for the regulation of DNA replication.     

Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens

A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.     

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.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

Characterization of a retrovirus shuttle vector capable of either proviral integration or extrachromosomal replication in mouse cells.

A retrovirus shuttle vector is described that contains the dominant selectable neo gene which confers resistance to kanamycin in bacteria and to the drug G418 in animal cells. The bacterial supF gene and the origins of DNA replication from polyomavirus and the ColE1 replicon also have been included in this vector. Infection of normal rodent cells results in single-copy proviral integration, whereas infection of mouse (MOP) cells expressing polyoma large T antigen results in extrachromosomal replication of the DNA form of the virus. The copy number of the extrachromosomal circles in MOP cells varies from 0 to 100 copies per cell. G418-resistant MOP cells lose their drug-resistant phenotype after passage under nonselective conditions, suggesting that maintenance of the extrachromosomal circles is unstable. The extrachromosomal form of the virus can be recovered as plasmids in Escherichia coli. Two-thirds of the circles analyzed were found to be structurally intact. The others have undergone rearrangements including deletions and insertions. The bacterial supF gene was found to be intact in the majority of recovered plasmids. The data presented here suggest that these retroviruses should be useful as gene transfer vectors for animal cells in culture or in vivo.     

Dilution enhancement of COS cell expression cloning

To search for an efficient expression cloning method, we mixed plasmid pmDATsv, which contains the mouse dopamine transporter (mDAT) cDNA, with a large amount of another plasmid prGlyTsv to mimic the situation of a cDNA library and examined COS cell expression. Both plasmids have an SV40 replication origin and thus will be replicated to high copy numbers in COS cells. After transfecting COS-7 cells with pmDATsv/prGlyTsv mixture at 1/1000 ratio, we could not detect any cells expressing strong mDAT activity. In contrast, when prGlyTsv was replaced by prSERTsk (no SV40 origin) in the transfection mixture, we observed hundreds of cells expressing strong mDAT activity. The results suggested that in many cells low mDAT expression was not due to the lack of pmDATsv plasmid but due to the presence of large numbers of replicable prGlyTsv. Analysis with a mathematical model suggests that diluting cDNA libraries with other plasmids without the SV40 origin should improve the detection of COS cells expressing target cDNAs. We tested this conclusion with pmDATsv/prGlyTsv mixture. When the mixture at 1/1000 ratio was diluted with prSERTsk and used for transfection, we could now easily detect cells expressing strong mDAT activity.     

Mechanism of interferon action. Expression of vesicular stomatitis virus G gene in transfected COS cells is inhibited by interferon at the level of protein synthesis

The effect of interferon on the expression of the vesicular stomatitis virus glycoprotein G gene was examined in simian COS cells transfected with the expression vector pSVGL containing the G gene under the control of the SV40 late promoter. When COS cells were treated with interferon 24 h after transfection, the synthesis of vesicular stomatitis virus G protein was inhibited by about 80% as compared to that in untreated controls. By contrast, under the same conditions, neither the plasmid copy number nor the G gene mRNA levels were detectably affected by interferon treatment. Likewise, the synthesis of simian virus 40 large T-antigen was not inhibited by interferon treatment of transfected COS cells even though the synthesis of vesicular stomatitis virus G protein was markedly inhibited. The residual G protein synthesized in transfected, interferon-treated COS cells appeared to be normally glycosylated.     

Transformation of human epidermal cells by transfection with plasmid containing simian virus 40 DNA linked to a neomycin gene in a defined medium

A human epidermal cell culture was transformed by transfection with a recombinant plasmid containing simian virus 40 DNA with a deletion at the origin and an antibiotic (neomycin or G418) marker. A calcium phosphate-mediated DNA transfection method was optimized for introducing exogenous DNA into cells maintained in a fully defined medium. The transformed cells were propagated for more than 200 population doublings and did not appear to go through a "crisis" period. The growth characteristics of the transformed cells were similar to those of untransformed cells. Major keratins synthesized in the transformed cells were similar to those found in normal epidermal cells. Transformed cells initially transfected with the recombinant plasmid could be propagated for more than 30 passages. Actively growing cells could then be repeatedly selected from cell populations based upon their neomycin (G418)-resistant phenotype for at least another 30 passages. Simian virus 40 T-antigen and extrachromosomal DNA containing plasmid- and SV40-specific DNA sequences were detected in the transformed cells. Because of their nononcogenic phenotype and defined growth requirements, the transformed cells provide a model for examining structural changes during cell proliferation and differentiation, and for exploring the multistage carcinogenesis of human epithelial cells.     

A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E. coli cells

We describe the construction of a bovine papilloma virus-based vector (pCGBPV9) which contains a dominant selectable marker and replicates autonomously in both mouse and Escherichia coli cells. This vector contains the complete bovine papilloma virus genome, a ColE1 replication origin and a dominant selectable marker conferring resistance to kanamycin in bacteria and G418 in eukaryotic cells. A high number of G418R colonies are obtained after transfer of pCGBPV9 into mouse C127 cells. These G418R colonies contain vector DNA which replicates autonomously at approximately 10-30 copies per cell. The molecules are in most cases unrearranged and can be rescued into E. coli cells by bacterial transformation.     

Amplification of Epstein-Barr virus-based shuttle vectors by ultraviolet light in human cells.

In order to approach the mechanism of gene amplification, we have developed a model system in human cells based on the use of episomally-replicating shuttle vectors. Shuttle vectors carrying the replication origin of the Epstein-Barr virus can be stably maintained in human cells. These vectors replicate as an episome with a low copy number. We also constructed hybrid plasmids containing both the EBV and the SV40 replication origins. These molecules are able to replicate episomally either like an EBV vector or like SV40 if the SV40 large T antigen is provided at the same time. UV irradiation of both human adenovirus transformed 293 or SV40-transformed MRC5 host cells leads to vector amplification whatever the type of replication origin used for the episomal maintenance. Our result clearly shows that the EBV latent replication origin (OriP), in the presence of the Epstein-Barr nuclear antigen-1 (EBNA-1) and the SV40 large T antigen, is sensitive to over-replication in UV-irradiated human cells. Since the UV doses were small enough to induce very little damage, if any, on the plasmid sequences, this amplification should be mediated through a cellular factor acting in trans. The interest in using shuttle vectors for this kind of study lays in the easy analysis of the amplified vectors in rescued bacterial colonies. The accuracy of the amplification process can be monitored by studying restriction maps of individual plasmid molecules or more precisely the integrity of a target gene, such as the lacZ' sequence, carried by our vectors.     

An inducible eukaryotic host-vector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a thermolabile large T antigen

We have taken advantage of the inherent instability of integrated polyoma (Py) DNA sequences in the presence of a functional viral large T antigen (LT) to develop a eukaryotic host-vector system where copy number is controlled by temperature. A mouse cell line WOP32-4, that constitutively expresses a temperature sensitive (ts) LT, was transfected with plasmids containing the Py origin of DNA replication (ori) and either a neomycin-resistance gene (neo) or chloramphenicol acetyl transferase gene (cat) linked to the Py late promoter. Stable transformants were selected at 39 degrees C, the non-permissive temperature for the ts LT function. Upon shift to 33 degrees C, the resident Py sequences present in the WOP32-4 cells cannot excise due to an ori deletion. However, excision of the transfected plasmid molecules and subsequent extrachromosomal replication occur at high rates leading in some cases to the production of 1000-2000 copies per cell (average) of the plasmid. Proportional increases in either neo-specific mRNA or CAT activity were also observed. In situ hybridization for one cell line indicated that about 20% of temperature-shifted cells contained amplified plasmid DNA.     

Negative control of DNA replication in composite SV40-bovine papilloma virus plasmids

To identify DNA sequences that function in the control of DNA replication, we designed a hybrid replicon consisting of linked SV40 and BPV DNA sequences. In the composite SV40-BPV plasmid negative control encoded by BPV is dominant over the uncontrolled replication encoded by the positive factor, SV40 T antigen. Using a transient replication assay, we show that replication control requires three BPV elements. Two cis-acting sequences are closely linked to BPV replication origins. A third trans-acting element is encoded within the 5' part of the BPV E1 open reading frame (ORF) and is separable from the positive replication factor encoded within the 3' part of the same ORF. The controlled replication of SV-BPV composite replicons has enabled us to create permanent COS cell lines that stably maintain these plasmids as episomes.     

Intermolecular plasmid recombination in fibroblasts from humans with DNA damage-processing defects

We have evaluated the ability of immortalized human fibroblasts to recombine transfected plasmid DNA. A number of cell lines from normal individuals and from patients with DNA damage-processing defects were examined. Two plasmid recombination substrates were derived from pSV2neo and contained nonoverlapping deletions in the aminoglycoside phosphotransferase II gene. Intermolecular recombination was assessed by two methods after cotransfection. In a short-term, extrachromosomal recombination assay, low molecular weight DNA was extracted from the human cells 48 h after transfection, and recombinant plasmids were detected by transformation into appropriate indicator bacteria. In a long-term stable recombination assay the fibroblasts were cotransfected and G418-resistant colonies allowed to form. By the former assay all but two cultures were recombination-proficient, whereas all were recombination-proficient by the latter assay. The efficiency of transfection of human cells with plasmids appears to be a major variable affecting recombination. Recombination can be stimulated by uv irradiation of plasmid DNA prior to transfection. Cells from patients with Fanconi anemia, ataxia telangiectasia, and xeroderma pigmentosum complementation groups A, C, D, E, and G are not defective at intermolecular plasmid recombination.