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.     

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.     

Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration.     

Establishment and maintenance of stable spatial patterns in lacZ fusion transformants of Polysphondylium pallidum.

Polysphondylium pallidum cells were transformed with a construct containing the Dictyostelium discoideum ecmA promoter fused to a lacZ reporter gene. Two stably transformed lines, one in which beta-galactosidase (beta-gal) is expressed in apical cells of the fruiting body (p63/2.1), and one in which it is expressed in basal cells (p63/D), have enabled us to infer how cells move during aggregation and culmination. Several types of cell movement proposed to occur during slime mold culmination, such as random cell mixing and global cell circulation, can be ruled out on the basis of our observations. Cells of the two transformant lines express beta-gal very early in development. In both cases, stained cells are randomly scattered in a starving population. By mid to late aggregation, characteristic spatial patterns emerge. Marked cells of p63/2.1 are found predominantly at tips of tight aggregates; those of p63/D accumulate at the periphery. These patterns are conserved throughout culmination, showing that marked cells maintain their relative positions within the multicellular mass following aggregation. Neither the apical nor the basal pattern appears to be regulated within the primary sorogen by de novo gene expression or by cell sorting as whorls are formed. However, marked cells within a whorl re-establish the original pattern in secondary sorogens. This must be achieved by cell migration, since beta-gal is not re-expressed.     

B族G蛋白偶联受体PAC1可调控表达体系的建立

PAC1是神经肽垂体腺苷酸环化酶激活多肽(Pituitary adenylate cyclase activating polypeptide,PACAP)的特异受体,属于B族G蛋白偶联受体,介导PACAP的神经递质、神经调质、神经保护、抗神经损伤及调控神经再生等功能,PAC1高表达和神经损伤、肿瘤等生理病理过程密切相关。为了深入了解PAC1的功能,构建PAC1可调控表达的细胞系,通过优化的四环素控制表达系统实现PAC1在中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞的强力霉素(doxycycline,Dox)依赖的可控表达。首先通过双酶切将编码PAC1和增强型黄色荧光蛋白(EYFP,enhanced yellow fluorescent protein)的融和基因PAC1-EYFP克隆到pTRE-Tight载体上,获得重组载体pTRE-PAC1-EYFP;基因测序鉴定正确后将新型的四环素调节元件载体pTet-on advanced和反应元件载体pTRE-PAC1-EYFP分别转入CHO细胞中,G418和潮霉素(Hygromycin)双抗筛选阳性克隆PAC1-Tet-CHO,使用梯度浓度四环素类似物强力霉素Dox诱导PAC1-EYFP表达,48 h后检测受体表达水平,并通过MTT法检测不同PAC1表达水平的细胞增殖活性。荧光检测和Western印迹结果显示,成功获得了具有良好诱导性的Dox依赖的PAC1可控表达的细胞系,这些细胞株在传10代后仍能稳定地可控表达PAC1。MTT结果显示PAC1表达水平越高,细胞增殖活性越强。成功所构建的Dox依赖的PAC1可控表达细胞系,为PAC1的生物学功能的深入研究奠定了基础。     

可调控表达甲型流感病毒M2蛋白的哺乳动物细胞系的建立与鉴定

甲型流感病毒M2蛋白是一种具有离子通道功能的跨膜蛋白,其氨基酸序列非常保守,可用于流感通用疫苗的研究。为了构建可调控的稳定表达甲型流感病毒M2蛋白的哺乳动物细胞系,首先应用PCR方法从含有流感病毒PR8株第七节段全长基因的质粒中扩增得到M2基因。将该片段亚克隆到真核表达载体pcDNA5/FRT/TO上,用BamHⅠ和NotⅠ双酶切鉴定正确后将重组质粒与表达Flp重组酶的pOG44质粒共转染Flp-In T-REx-293细胞,使目的基因整合到宿主细胞染色体。筛选具有Hygromycin B抗性的细胞株。在该细胞的培养基中加入四环素以诱导目的基因表达,48 h后通过间接免疫荧光方法检测到M2蛋白的表达。共得到16株高表达M2蛋白的重组细胞株,这些细胞株在传10代后仍能稳定表达目的蛋白。未加四环素诱导的细胞没有检测到M2蛋白,说明四环素调控系统严格控制着目的基因的表达。今后,该细胞系可用于流感病毒M2蛋白的功能研究、流感候选疫苗的免疫学评价以及流感病毒减毒活疫苗的研制。     

Establishment of tsA58 transgenic rats as a source of conditionally immortalized cell lines with differentiated functions.

To isolate a variety of rat cell lines with differentiated functions, we developed transgenic rat lines that ubiquitously express the temperature-sensitive large T-antigen gene of the simian virus 40 (SV40) tsA58 mutant under the control of the SV40 large T-antigen promoter. These rats might be advantageous for simultaneously establishing cell lines from different tissues of rats with the same genetic origin. The transgenic rat lines transmit a functional copy of the transgene and were bred with sib mating to generate the homozygous transgene. The established cell lines from this transgenic rat had temperature dependent growth and retained some of the differentiated functions of each particular tissue, and were useful as a ready source of novel conditionally immortalized cell lines. The possible use and perspectives of these transgenic cell lines are discussed.     

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.     

Tetracycline-Inducible System for Regulation of Skeletal Muscle-Specific Gene Expression in Transgenic Mice

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.     

受强力霉素调节的自杀基因表达载体系统的构建和在乳腺癌细胞株(MCF-7)的表达

自杀基因治疗是恶性肿瘤基因治疗中最常用的途径之一,以递转录病毒载体-pRevTRE为基础进行载体构建,首先使用PCR技术对单纯疱疹病毒胸苷激酶基因（HSVtk)进行扩增,将HSVtk基因插入到pRe-vTRE,形成重组载体pRevTRE/HSVtk,用磷酸钙共沉淀法,经过两轮转染,分别将pRevTRE/HSVtk和pRevTet-On质粒导入乳腺癌细胞株（MCF-7）经过潮霉素B（HygromycinB)和G418筛选,建立了一株稳定的受四环素衍生物-强力霉素（Doxycycline,Dox)调控,表达HSVtk基因产物的人乳腺癌细胞株MCF/TRE/tk/Tet-On,HSVtk基因表达产物可以将无毒性的药物前体Ganciclovir(GCV)转变成一种有毒的代谢产物,从而杀死乳腺癌细胞株（MCF-7）,达到基因治疗的目的。     

A Novel Piggybac Transposon Inducible Expression System Identifies a Role for Akt Signalling in Primordial Germ Cell Migration

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.     

Silencing of mammalian genes by tetracycline-inducible shRNA expression

Conditional gene silencing in mammalian cells, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying gene function, particularly if the gene is essential for cell survival or development. Here we describe a simple and rapid protocol for the generation of tetracycline (Tet)-inducible vectors that express shRNAs in a time- and dosage-dependent manner. Tet-operator (TetO) sequences responsive to occupation by the Tet-repressor (TetR) were inserted at alternative positions within the wild-type H1 promoter and cloned into a eukaryotic expression vector. Additional cloning sites downstream of the promoter enable the insertion of shRNA sequences. This Tet-inducible shRNA expression system can be used for both transient and stable RNA interference (RNAi) approaches to control gene function in a spatiotemporal fashion. The entire protocol (preparation of constructs, generation of stable cell lines and functional analysis) can be completed in 3 months.     

P-glycoprotein mediates resistance to A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-n-methyluronamide in human leukemia cells

We studied effects of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) on apoptosis induction in the K562/Dox cell line, which overexpressed P-glycoprotein (P-gp, ABCB1, MDR1). We found that the K562/Dox cell line was significantly more resistant to Cl-IB-MECA than the maternal cell line K562, which did not express P-gp. Although both cell lines expressed the A3 adenosine receptor (A3AR), cytotoxic effects of Cl-IB-MECA were not prevented by its selective antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate). Analysis of cell extracts revealed that the intracellular level of Cl-IB-MECA was significantly lower in the K562/Dox cell line than in the maternal cell line K562. The downregulation of P-gp expression using shRNA targeting ABCB1 gene led to increased intracellular level of Cl-IB-MECA and restored cell sensitivity to this drug. Similarly, valspodar (PSC-833), a specific inhibitor of P-gp, restored sensitivity of the K562/Dox cell line to Cl-IB-MECA with concomitant increase of intracellular level of Cl-IB-MECA in the resistant cell line, while it affected cytotoxicity of Cl-IB-MECA in the sensitive cell line only marginally. An enzyme based assay provided evidence for interaction of P-gp with Cl-IB-MECA. We further observed that cytotoxic effects of Cl-IB-MECA could be augmented by activation of extrinsic cell death pathway by Apo-2L (TRAIL) but not FasL or TNF-α. Our results revealed that Cl-IB-MECA induced an increase in expression of TRAIL receptors in K562 cells, which could sensitize cells to apoptosis induction via an extrinsic cell death pathway. Importantly, these effects were inversely related to P-gp expression. In addition, MRS1523 did not affect Cl-IB-MECA induced expression of TRAIL receptors.     

Developing tTA Transgenic Rats for Inducible and Reversible Gene Expression

To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 μg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 μg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases.