Retroviral delivery of the ecdysone-regulatable gene expression system

We describe a set of Moloney Murine Leukemia Virus (MoMLV)-based replication-defective retroviral vectors for delivery of the ecdysone-inducible system into mammalian cells. The vector pFB-ERV contains a tricistronic CMV expression cassette from which the ecdysone receptor proteins RXR and VgEcR are expressed, with the neo-resistance marker expressed as the third open reading frame (ORF). The inducible vector pCFB-EGSH contains an ecdysone-inducible expression cassette inserted between the viral LTRs in the antisense orientation relative to that for the viral promoter. Potential interference from the proviral 5' LTR is obviated due to a SIN deletion in the 3' LTR. When used together, induction ratios of over 1000-fold were achieved in NIH3T3 cells using firefly luciferase as a reporter.     

Reporter-linked monitoring of transgene expression in living cells using the ecdysone-inducible promoter system

Inducible promoter systems such as the ecdysone-inducible system or the tetracycline-regulated expression systems have proven to be powerful tools in studying gene function. In practice, such systems have met with the difficulty that either the vector expressing the transactivator gene or the vector carrying the response element are frequently silenced by flanking genomic sequences after stable integration. In order to identify those cells in a heterogeneous population in which a transgene is expressed from an ecdysone-inducible promoter, we have created the vector p2ER-EGFP/mcs that contains two ecdysone-inducible expression cassettes in tandem. Using two reporter genes, lacZ and green fluorescent protein (EGFP), we demonstrate that the expression of both genes can be co-induced from a very low baseline in CHO cells expressing the modified ecdysone receptor and the retinoid X receptor. The expression of EGFP and lacZ from vector p2ER-EGFP/lacZ follows the same Muristerone A concentration-dependence as that of EGFP from vector pER-EGFP, indicating that the juxtaposition of the two inducible promoters in vector p2ER-EGFP/mcs does not cause cross interference between them. We suggest that this modification of the ecdysone-inducible promoter system will allow for the visual control of the induced expression of other genes by Muristerone A.     

IPTG-inducible episomal expression system for exogenous genes in primate cells

An isopropyl beta-D-thiogalactopyranoside (IPTG)-inducible episomal expression system has been established for the human breast carcinoma cell line MCF7. This two-component system includes: (i) a primate cell-specific episomal vector, pEpiLac, that contains an IPTG-inducible promoter and (ii) a cell line derived from MCF7, MCF7/LAP5, which expresses the IPTG-dependent transactivator LAP267. Treatment of MCF7/LAP5 cells with IPTG results in efficient inducible expression of exogenous genes from the inducible promoter in pEpiLac. Up to 300-fold induction can be observed when luciferase is used as a reporter. Inducible expression of the p27KIP1 cyclin-dependent kinase (CDK) inhibitor, the orphan nuclear receptor Nur77 and the angiogenic inducer Cyr61 has also been demonstrated. Expression of the exogenous gene is promptly halted on removal of IPTG. Moreover, the episomal vector can be stably maintained in and easily recovered from MCF7/LAP5 cells. Taken together, this inducible expression system should be applicable for the regulated expression of exogenous genes, especially growth inhibitory or cytotoxic genes, in cells of primate origin.     

Development of a ponasterone A-inducible gene expression system for application in cultured skeletal muscle cells

The goal of this study was to develop an inducible gene expression system to assess functions of specific proteins in differentiated cultured skeletal muscle. We utilized and modified the ecdysone inducible system because others have used this system to express exogenous genes in vitro and in transgenic animals. A limitation of the commercially-available ecdysone system is its constitutive expression in all tissues. Hence, its application in vivo would result in expression of a cloned gene in undifferentiated and differentiated tissues. To target its expression to muscle, we removed the constitutively-active CMV promoter of pVgRXR and replaced it with a skeletal muscle alpha-actin promoter so that the regulatory features of the system would be expressed in differentiated muscle cells. We transfected our newly designed expression system into L8 muscle myoblasts and established stable cell lines via antibiotic selection. We determined that reporter gene activity was induced by ponasterone A in myotubes, a differentiated muscle phenotype, but not in myoblasts (undifferentiated cells). This proved the validity of the concept of an inducible muscle-specific expression system. We then determined that beta-galactosidase expression was dependent upon the dose of ponasterone A and duration of exposure to inducer. This creates potential to regulate both the level of expression and duration of expression of a cloned gene in differentiated muscle.     

蜕皮激素诱导MIIP基因的表达对肝癌细胞增殖迁移的影响

目的:探讨蜕皮激素诱导迁移侵袭抑制蛋白基因(migration and invasion inhibitory protein,MIIP)基因的表达对肝癌细胞SK-Hep-1增殖和迁移能力的影响。方法:采用PCR扩增MIIP基因,连入T载体测序正确后,插入到蜕皮激素可诱导真核表达载体pIND中。将pIND-MIIP和含蜕皮激素受体基因的表达载体pVgRXR以脂质体转染法转染到SK-Hep-1中,经G418和Zeocin双抗生素筛选获得稳定转染细胞株。蜕皮激素Ponasterone A诱导后,采用Western blot检测MIIP表达,CCK-8实验检测细胞增殖,划痕实验检测细胞迁移。结果:蜕皮激素诱导MIIP表达上调后,SK-Hep-1细胞的增殖能力显著下降(P0.05),细胞的迁移能力明显减弱。结论:建立了MIIP基因可诱导表达系统,证实在肝癌细胞SK-Hep-1中MIIP过表达可抑制细胞的增殖及迁移能力。     

A bicistronic, Ubiquitin-10 promoter-based vector cassette for transient transformation and functional analysis of membrane transport demonstrates the utility of quantitative voltage clamp studies on intact Arabidopsis root epidermis

To date the use of fluorescent reporter constructs in analysing membrane transport has been limited primarily to cell lines expressing stably either the tagged transporter protein(s) or markers to identify lineages of interest. Strategies for transient expression have yet to be exploited in transport analysis, despite their wide application in cellular imaging studies. Here we describe a Gateway-compatible, bicistronic vector, incorporating the constitutive Ubiqutin-10 gene promoter of Arabidopsis that gives prolonged expression after transient transformation and enables fluorescence marking of cells without a fusion construct. We show that Arabidopsis root epidermal cells are readily transformed by co-cultivation with Agrobacterium and are tractable for quantitative electrophysiological analysis. As a proof of principle, we transiently transformed Arabidopsis with the bicistronic vector carrying GFP as the fluorescent marker and, separately, the integral plasma membrane protein SYP121 essential for the inward K+ channel current. We demonstrate that transient expression of SYP121 in syp121 mutant plants is sufficient to rescue the K+ current in vivo. The combination of transient expression and use of the bicistronic vector promises significant advantages for studies of membrane transport and nutrient acquisition in roots.     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

Tightly Regulated and Homogeneous Transgene Expression in Human Adipose-Derived Mesenchymal Stem Cells by Lentivirus with Tet-Off System

Genetic modification of human adipose tissue–derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 α (EF-1α) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.     

Inducible expression and pharmacology of recombinant NMDA receptors,composed of rat NR1a/NR2B subunits

An ecdysone-inducible mammalian expression system was used to study expression of recombinant N-methyl-D-aspartate (NMDA) receptors. Human embryonic kidney (HEK) 293 cells expressing the regulatory vector pVgRXR (EcR 293 cells) were transfected with rat NR1a and NR2B cDNAs using the inducible vector pIND (Invitrogen). Inducible expression of the NR2B subunit in cell clone designated EcR/rNR1a2B was investigated using quantitative RT-PCR and flow cytometry based immunocytochemical methods. The mRNA level of the NR2B subunits in EcR/rNRa2B cells was dependent on the concentration of the ecdysone analogue inducing agent, muristerone A (MuA). Similarly, NR2B subunit protein expression was higher in cells pre-treated with the inducing agent. Functionally active NMDA receptors were also detected in EcR/rNR1a2B cells after MuA induction. In presence of the inducing factor, NMDA-evoked ion currents as well as increase in cytoplasmic calcium-concentrations were measured using whole-cell patch clamp and fluorometric calcium measuring techniques. The pharmacological profile of the expressed NMDA receptors was characterised by comparing the inhibitory activity of several NR2B subunit selective NMDA antagonists in EcR/rNR1a2B cells with that observed in primary cultures of rat cortical neurones. Whereas the efficacies of the NR2B subunit selective NMDA antagonists were similar in EcR/rNR1a2B cells and in neurones, their maximal inhibitory effects were significantly higher in cells expressing NR1a/NR2B recombinant receptors. This study demonstrates that recombinant NMDA receptors can be expressed in an inducible way in non-neuronal cell lines using the ecdysone-inducible mammalian expression system. Such cell lines can be suitable tools in high throughput functional screening for potential subtype selective modulators of the NMDA receptor.     

CMV与T7启动子对仙台病毒微小基因组拯救效率的比较

构建一种以分泌型荧光素酶基因(Gluc)作为报告基因的仙台病毒BB1株微小基因组质粒,比较了CMV启动子与T7启动子对仙台病毒微小基因组的拯救效率。首先设计并合成锤头状核酶序列,仙台病毒trailer、L基因非编码区、N基因非编码区和leader序列以及丁型肝炎病毒核酶序列,插入含有CMV和T7双启动子的质粒pVAX1中,获得仙台微小基因组的通用型载体pVAX-miniSeV。将Gluc基因插入pVAX-miniSeV中,分别获得正向插入的仙台病毒微小基因组载体pVAX-miniSeV-Gluc(+)和反向插入的pVAX-miniSeV-Gluc(-)。用pVAX-miniSeV-Gluc(+)转染BHK21细胞能在上清中检测到高水平的Gluc活性,表明其中的CMV启动子具有正常转录功能。将pVAX-miniSeVGluc(-)和仙台病毒N、P、L蛋白表达质粒共转染BSR T7/5细胞(稳定表达T7RNA聚合酶的BHK-21细胞)检测到Gluc的高效表达,表明pVAX-miniSeV-Gluc(-)能够被有效拯救;但在BHK-21细胞中却未检测到Gluc的有效表达,提示该载体中的CMV启动子对仙台病毒微小基因组的拯救效率可能没有明显作用。为了进一步了解CMV与T7启动子各自对于仙台病毒微小基因组拯救的作用,本研究又构建了单独含有CMV或T7启动子的仙台病毒微小基因组载体pCMV-miniSeV-Gluc(-)和pT7-miniSeV-Gluc(-)。将这两种载体和仙台病毒N、P、L蛋白表达质粒分别共转染BSR T7/5细胞,结果pT7-miniSeV-Gluc(-)共转染组检测到了Gluc的高效表达,而pCMV-miniSeV-Gluc(-)共转染组未检测到,证实了通用型载体pVAX-miniSeV中仅T7启动子对仙台病毒微小基因组的拯救起了关键作用,而CMV启动子作用不明显。本研究成功构建了一种通用型双启动子仙台病毒微小基因组载体pVAX-miniSeV,并证明了T7启动子系统对仙台病毒微小基因组拯救的关键作用。本研究为下一步构建仙台病毒全基因感染性克隆打下了基础。     

Ecdysone-inducible foreign gene expression in stably-transformed lepidopteran insect cells

Cultured cell lines that can be stably transformed with inducible gene constructs could prove extremely valuable for the continuous and economical production of recombinant proteins. Toward this goal, we have established 11 clones (designated NISES-BoMo-DK1 to 11) from a previously reported silkworm cell line, NISES-BoMo-DZ. Nine of these clonal lines showed a distinct morphological change. i.e., cell aggregation, in response to treatment with 1 microM 20-hydroxyecdysone (20E). DK10 cells transfected with various reporter assay plasmids under optimal conditions (i.e., 20-30% transfection efficiency) showed inducibility of gene expression by 20E. The 20E treatment of the prototypical DK10 cells resulted in a simultaneous, transient increase of the nuclear ecdysone (E) receptor levels. Further, this inducibility was also observed in a DK10 cell line stably transformed with the reporter plasmid that carries the hygromycin-resistance gene. This offers an opportunity to achieve efficient, continuous production of recombinant proteins. It could also allow high throughput screening for potential E agonists.     

Functional analysis of the transcriptional control regions of the copia transposable element

The introduction of copia-based vectors in Drosophila hydei cells results in their high-level transient expression and the subsequent establishment of stably transformed cell lines containing multiple copies of vector integrated into host genomic DNA. Using transformation frequency and transient expression analysis as assays of promoter strength, we have defined the regions of copia essential for expression. We find that the essential sequences reside within the long terminal repeat, but 3' to the site of initiation of copia RNA. Deletion of the consensus enhancer-like sequences from copia appears to have no effect on vector expression.     

Generation of a replication-competent,propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome

Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E(+) packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic TGEV E protein did not interfere with the rescue of infectious TGEV from full-length cDNA. Recombinant TGEV deficient in the nonessential 3a and 3b genes and the essential E gene (rTGEV-Delta3abDeltaE) was successfully rescued in these cell lines. rTGEV-Delta3abDeltaE reached high titers (10(7) PFU/ml) in baby hamster kidney cells expressing porcine aminopeptidase N (BHK-pAPN), the cellular receptor for TGEV, using Sindbis replicon and reached titers up to 5 x 10(5) PFU/ml in cells stably expressing E protein under the control of the CMV promoter. The virus titers were proportional to the E protein expression level. The rTGEV-Delta3abDeltaE virions produced in the packaging cell line showed the same morphology and stability under different pHs and temperatures as virus derived from the full-length rTGEV genome, although a delay in virus assembly was observed by electron microscopy and virus titration in the complementation system in relation to the wild-type virus. These viruses were stably grown for >10 passages in the E(+) packaging cell lines. The availability of packaging cell lines will significantly facilitate the production of safe TGEV-derived vectors for vaccination and possibly gene therapy.     

Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity, robust expression, and reduced size

The ecdysone receptor (EcR)-based gene regulation system is a tool for controlling gene expression. To improve the sensitivity of this system, we evaluated many two-hybrid format synthetic gene constructs in which the GAL4 DNA binding domain was fused to the ligand binding domain of the Choristoneura fumiferana EcR mutant V390I/Y410E (GEvy), and various activation domains--VP16, p53, p65, or E2F-i--were fused to the EF domains of chimeric human RXR. These gene switches were assayed in NIH3T3 cells, HEK293 cells, and in mouse quadriceps in the presence of the nonsteroidal inducer RG-115819 or GS-E. All of the two-hybrid format constructs had no or very low background in the "off" condition and high luciferase reporter gene expression levels in "on" conditions. Extremely high sensitivity was achieved, with EC50 values in the subnanomolar range and with maximal induction at 10 nM RG-115819. Co-expression of both receptor genes with encephalomyocarditis virus (EMCV) or eIF4G internal ribosome entry site (IRES) sequences gave robust induction levels. To reduce the size of the switch construct, we tested single receptor formats, in which any of 14 different activation domains were fused to GEvy. We identified several switches with acceptable levels of basal and maximal induction levels. The gene switches described here provide receptor configuration options suitable for gene function studies, therapeutic protein production in cell culture, transgenic mouse models, and gene/cell therapy.