Effect of DNA damage on the expression of the chloramphenicol acetyltransferase gene after transfection into diploid human fibroblasts.

The activity of the chloramphenicol acetyltransferase (cat) gene after transfection into human fibroblasts has been measured following treatment of the plasmid pRSVcat with either restriction enzymes or ultraviolet light. Restriction enzymes producing single cuts in the plasmid inactivated the expression of the cat gene whether the enzymes cut the plasmid inside the coding region of the gene or several kilobases away from the gene. Ultraviolet light produced a dose-dependent inactivation of the gene. The inactivation curve was steeper if the recipient cell strain was derived from a patient with xeroderma pigmentosum. The findings with this transient expression system contrast with previously reported results of experiments using plasmids which transform cells stably by integrating into the cellular genomic DNA.     

Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.     

Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding, cell uptake, expression, and integration into host chromosomes of DNA in the mammalian cell.

Neumann and coworkers (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO J. 1:841-845) have shown that the efficiency of pulsed electric field (PEF)-induced DNA transfection of mouse L-cells by the thymidine kinase gene is several times higher for the linear DNA than for the closed circular DNA. Transfection of Escherichia coli bacteria by several plasmids indicates that the transfection efficiency was much higher for the closed circular/supercoiled (sc-) and circular/relaxed (cr-) DNA than for the linearized (In-) DNA (Xie, T. D., L. Sun, H. G. Zhao, J. A. Fuchs, and T. Y. Tsong. 1992. Biophys. J. 63:1026-1031). To resolve these conflicting observations, we have systematically examined electrotransfection of NIH3T3 mouse fibroblast by the plasmids, pRSVcat, pRSVneo, and pRSVgpt. Mg(2+)-facilitated surface binding of DNA before, and DNA uptake by 3T3 cells after treatment with PEF, were monitored by 3H-labeled plasmids. Transfection efficiency was evaluated by both the transient expression of chloramphenicol acetyltransferase (cat) activity 2-3 days after, and the permanent expression of neomycin phosphotransferase (neo) and xanthine-guanine phosphoribosyltransferase (gpt) genes in the transformants 2 weeks after the PEF treatment. Our results indicate that cell surface binding and PEF-induced cell uptake of DNA did not depend on the topology of DNA. However, both the transient and the permanent expression of the plasmids were three to five times more efficient for the cr-DNA and the sc-DNA than for the in-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication

We have examined the activation of the SV40 late promoter by inserting the late promoter and the viral origin of replication into chloramphenicol acetyltransferase (CAT) transient expression vectors. Very little late promoter activity was detected in CV-1 cells, compared with high activity in COS cells, in which replication occurs due to endogenous T antigen. Nonreplicative counterparts of these plasmids, containing a mutated origin of replication, produced significantly more late promoter activity in COS cells than any of the plasmids in CV-1 cells. When plasmids were cotransfected into CV-1 cells with a plasmid that supplies T antigen, the nonreplicative plasmid displayed 30% of the activity of the replicative plasmid. Using mutant T antigens unable to replicate viral DNA, late promoter activation occurred only with mutant T antigens that retain DNA binding activity. These results demonstrate that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication.     

Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.     

Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.     

人U6基因POI Ⅲ启动子表达反义VEGF cDNA抑制SMMC-7721肝癌细胞VEGF表达的观察

 由 RNA聚合酶启动子在细胞内转录高浓度反义 RNA是抑制靶蛋白的一种有效手段 ,有报道 POI 启动子转录小分子 RNA存在效率不高 ,带有较多的非特异性序列等缺点 ,为了克服这些问题 ,表达反义 VEGF RNA的人 U6基因表达盒对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制作用进行了研究 .首先 PCR扩增 2 0 0 bp VEGF c DNA以正、反向插入人 U6 sn RNA.通过测序证实反向插入的正确性 .采用细胞原位杂交 ,RNA酶保护分析 ,Northern印迹来证实反义 RNA表达的情况 ,利用 RT- PCR方法研究了其对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制效果 .细胞原位杂交结果显示 U6启动子转录产物主要分布于细胞核内 ,细胞浆内亦有表达 ,RNA酶保护分析显示 U6基因 POI 启动子能高表达所需大小反义 RNA,Northern印迹结果显示脂质体 lipo-fectamine介导含 U6基因 POI 启动子的质粒转染人肝癌细胞株 SMMC- 772 1后 1 2 h即有表达且可持续表达 6 d. RT- PCR证实 U6基因 POI 启动子转录的反义 VEGF RNA能有效抑制SMMC- 772 1细胞 VEGF的 m RNA表达 .已有的研究结果揭示 POI 启动子是反义基因治疗中一种好的选择     

High-efficiency gene transfection by in situ electroporation of cultured cells

It is demonstrated in this study that high-efficiency gene transfection can be obtained by directly electroporating cultured mammalian cells in their attached state using a pulsed radio-frequency (RF) electric field. A plasmid DNA containing the reporter gene beta-gal was introduced into COS-M6 cells and CV-1 cells using this in situ electroporation method. At the optimal electric field strength (1.2 kV/cm), we found that over 80% of the M6 cells took up and expressed the beta-gal gene with a cell survival rate of about 50%. In contrast, the transfection efficiency was less than 20% when the M6 cells were electroporated in suspension. It was shown that CV-1 cells could also be electroporated highly efficiently using the in situ method. Furthermore, we have measured the time required to express the beta-gal gene after the plasmid DNA was introduced. We found that the percentage of cells expressing beta-gal reached a peak value about 10 h after electroporation. This time-course was the same for both attached and suspended cells, suggesting that the observed difference in transfection efficiency was mainly the result of effects of the detachment treatment on the electroporation process rather than on the gene expression.     

Structure of recombinant plasmids containing synthetic human foetal globin gene sequences

Summary In vitro synthesized duplex DNA complementary to human foetal globin messenger RNA was integrated into bacterial plasmids and amplified by transformation of Escherichia coli. Recombinants carrying globin DNA were identified by hybridization of foetal globin messenger RNA to bacterial DNA in situ and by liquid hybridization of purified plasmids to specific globin complementary DNA probes. Heteroduplex mapping revealed either a simple insertion loop at the position of the EcoRI site of the parental plasmid or substitution loops due to insertion of globin DNA sequences combined with deletions of the parental plasmid DNA. We provide evidence to suggest that these deletions are the result of a site-specific nicking activity of the EcoRI preparations used in the formation of recombinant plasmids.     

Vectors bicistronically linking a gene of interest to the SV40 large T antigen in combination with the SV40 origin of replication enhance transient protein expression and luciferase reporter activity

The simian virus 40 large T antigen (SVLT) induces replication of plasmids bearing the SV40 origin of replication (SV40 ori) within mammalian cells. The internal ribosomal entry site (IRES) is an element that allows for the cotranslation of proteins from one polycistronic mRNA. Through the combination of these elements, IRES-dependent coexpression of a protein of interest and the SVLT, either constitutive or regulated, on plasmids bearing the SV40 ori generates a positive feedback loop, resulting in enhanced expression. A vector linking red fluorescent protein (RFP) to the IRES-SVLT element enhances fluorescence ~10-fold over that demonstrated from a vector lacking this element. In transfection-resistant CV-1 cells, the RFP-IRES-SVLT vector substantially increases the number of cells expressing detectable levels of RFP. Furthermore, inclusion of the IRES-SVLT/SV40 ori elements in standard luciferase-based reporter gene constructs and associated effectors results in marked increases in luminescent output and sensitivity, using the β-catenin/TCF pathway and the mammalian two-hybrid assay as models. Ultimately, vector systems combining these well-established elements (IRES-SVLT/SV40 ori) will increase the utility of transient transfection for the production of recombinant proteins, the use of transfection-resistant cell lines, and the effectiveness of luciferase-based high-throughput screening assays.     

Efficient gene transfer by sequential treatment of mammalian cells with DEAE-dextran and deoxyribonucleic acid

A variation of the classical DEAE-dextran method of gene transfer was developed for efficient transfection of HeLa cells with plasmid DNA. A brief exposure of the cells to medium containing DEAE-dextran was found to be sufficient for subsequent uptake of pRSVcat and to be superior to cocultivation of the cells with DEAE-dextran plus DNA. This sequential method of gene transfer is nontoxic and yielded up to 60% of HeLa cells positive for a surface protein encoded by the transfected sequence. The implications of this sequential transfection technique regarding the mechanisms of gene transfer are discussed.     

T7启动子在哺乳类动物细胞中启动外源基因表达的研究

人低密度脂蛋白（LDL）受体基因cDNA和氯霉素已酞转移酶基因（CAT）及PolyA信号序列被克隆进pGEM4载体的T7噬茵体启动子下游,构建成质粒pT7LDLR和pT7CAT．两个重组质粒转化CHO细胞．PCR和CAT酶实验显示：两个基因被T7噬菌体启动子所启动．结果证实真核生物RNA聚合酶能够识别T7启动子,转录外源基因．常用的含有T7启动子的质粒可同时作为原核生物和真核生物的表达载体．     

Generation of glucocorticoid-responsive Moloney murine leukemia virus by insertion of regulatory sequences from murine mammary tumor virus into the long terminal repeat.

The glucocorticoid-regulatory sequences from the murine mammary tumor virus long terminal repeat (MMTV LTR) were introduced into the LTR of Moloney murine leukemia virus (M-MuLV) by recombinant DNA techniques. The site of insertion was in the M-MuLV LTR U3 region at -150 base pairs with respect to the RNA cap site. Infectious M-MuLVs carrying the altered LTRs (Mo + MMTV M-MuLVs) were recovered by transfection of proviral clones into NIH-3T3 cells. The Mo + MMTV M-MuLVs were hormonally responsive in that infection was 3 logs more efficient when performed in the presence of dexamethasone, irrespective of the orientation of the inserted MMTV sequences. However, even in the presence of hormone, the Mo + MMTV M-MuLVs were less infectious than wild-type M-MuLV. In contrast to the large effect on infectivity, dexamethasone induced virus-specific RNA levels in chronically Mo + MMTV M-MuLV-infected cells only two- to fourfold. Fusion plasmids between the altered LTRs and the bacterial chloramphenicol acetyltransferase gene allowed the investigation of LTR promoter strength by the transient chloramphenicol acetyltransferase expression assay. The chloramphenicol acetyltransferase assays indicated that the insertion of MMTV sequences into the M-MuLV LTR reduced promoter activity in the absence of glucocorticoids but that promoter activity could be induced two- to fivefold by dexamethasone. The Mo + MMTV M-MuLVs were also tested for the possibility that viral DNA synthesis or integration during initial infection was enhanced by dexamethasone. However, no significant difference was detected between cultures infected in the presence or absence of hormone. The insertion of MMTV sequences into an M-MuLV LTR deleted of its enhancer sequences did not yield infectious virus or active promoters, even in the presence of dexamethasone.