Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters

BACKGROUND: The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. METHODS: We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. RESULTS: We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. CONCLUSIONS: Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.     

Temporal and spatial control of transgene expression using a heat-inducible promoter in transgenic wheat

Constitutive promoters are widely used to functionally characterise plant genes in transgenic plants, but their lack of specificity and poor control over protein expression can be a major disadvantage. On the other hand, promoters that provide precise regulation of temporal or spatial transgene expression facilitate such studies by targeting over-expression or knockdown of target genes to specific tissues and/or at particular developmental stages. Here, we used the uidA (beta-glucuronidase, GUS) reporter gene to demonstrate that the barley Hvhsp17 gene promoter can be induced by heat treatment of 38-40 °C for 1-2 h in transgenic wheat. The GUS enzyme was expressed only in those tissues directly exposed to heat and not in neighbouring leaf tissues. The induction of HSP::GUS was demonstrated in all organs and tissues tested, but expression in older tissues was lower. Generally, proximal root sections showed less GUS activity than in root tips. This heat-inducible promoter provides the ability to investigate the function of candidate genes by overexpression or by down-regulation of target gene expression (for example by RNAi) in selected tissues or developmental stages of a transgenic plant, limited only by the ability to apply a heat shock to the selected tissues. It also allows the investigation of genes that would be lethal or reduce fertility if expressed constitutively.     

四环素诱导调控表达系统的研究与应用

四环素(Tet)诱导调控表达系统是在大肠杆菌Tn10转座子中特异的Tet抗性操纵子基础上建立起来的一种用于诱导基因表达的调控系统。经过近几年来的发展,衍生出了Tet-on、Tet-off两套调控系统;随着对Tet诱导调控表达系统的改进,其对基因表达调控的严谨性、诱导效率及安全性等方面逐步得到改善,并被广泛应用于基础研究及体内外的实验治疗研究。此外Tet诱导调控系统与其他诱导调控体系及新兴技术的联合应用,为疾病的基因治疗研究提供有力的手段。     

Tet-On systems for doxycycline-inducible gene expression

The Tet-On advanced inducible gene expression system in vitro is known for genarating robust expression of the desired gene in target cells. The system offers many advantages over other inducible mammalian gene expression systems, such as high specificity, high inducibility, and high absolute expression levels. In this study, the Tet-On advanced inducible gene expression system was applied to induce the expression of the trophoblast cell-surface antigen 2 (Trop-2) gene in vitro and explore the biological functions of Trop-2. 293/pTet-On-Advanced cell lines were generated, and a recombinant vector containing the Trop-2 gene was constructed and transfected into stable cell lines to improve Trop-2 protein expression. In the presence of doxycycline (DOX), the proliferation assay, transwell assay, and wound healing assay were performed to analyze the efficacy of Trop-2. The results showed that the Tet-On advanced inducible gene expression system was established successfully in the cell line 293, Trop-2 protein level in cells was significantly increased, and Trop-2 could enhance growth, migration, and aggression in the cell line 293. This study suggests that the Tet-On advanced inducible gene expression system can induce the expression of interest genes specifically and artificially in vitro and provides a viable and convenient platform for the study of gene function.     

Expression of inducible angiosperm promoters in a gymnosperm,Picea glauca (white spruce)

Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnospermPicea glauca (white spruce). Promoter expression was assayed in three different tissues capable ofin vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducibleArabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter--glucuronidase-nopaline synthase 3 fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the -glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.     

Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice

The human placental alkaline phosphatase (PLAP) gene was analysed for its utility as a histochemically detectable reporter gene in transgenic mice. A reporter gene was made by linking the PLAP structural gene to an enhancerpromoter element from the human -actin gene. This gene was inserted into the mouse genome by transfection of embryonic stem cells, and by microinjection of fertilized eggs. Histochemical staining showed that the transgene was uniformly expressed in four of four stable ES cell lines, and in all ten tissues examined from adult animals from five lines of transgenic mice. Non-transgenic cells did not stain. These results suggest that the human PLAP gene will be of utility in studies requiring phenotypic marking of cells in tissues of mice.     

One-step knockin for inducible expression in mouse embryonic stem cells

Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline (dox)-controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units, and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor, and money. Here, we describe a one-step Cre-mediated knockin system in mouse embryonic stem cells that positions the TA and inducible expression cassette to a single location. Using this system, we show dox-dependent regulation of eGFP at the DNA topoisomerase 3β promoter. Because Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.     

Inducible and neuron-specific gene expression in the adult mouse brain with the rtTA2S-M2 system

To achieve inducible and reversible gene expression in the adult mouse brain, we exploited an improved version of the tetracycline-controlled transactivator-based system (rtTA2(S)-M2, rtTA2 hereafter) and combined it with the forebrain-specific CaMKIIalpha promoter. Several independent lines of transgenic mice carrying the CaMKIIalpha promoter-rtTA2 gene were generated and examined for anatomical profile, doxycycline (dox)-dependence, time course, and reversibility of gene expression using several lacZ reporter lines. In two independent rtTA2-expressing lines, dox-treatment in the diet induced lacZ reporter expression in neurons of several forebrain structures including cortex, striatum, hippocampus, amygdala, and olfactory bulb. Gene expression was dose-dependent and was fully reversible. Further, a similar pattern of expression was obtained in three independent reporter lines, indicating the consistency of gene expression. Transgene expression could also be activated in the developing brain (P0) by dox-treatment of gestating females. These new rtTA2-expressing mice allowing inducible and reversible gene expression in the adult or developing forebrain represent useful models for future genetic studies of brain functions.     

The effect of MAR elements on variation in spatial and temporal regulation of transgene expression

The level of transgene expression often differs among independent transformants. This is generally ascribed to different integration sites of the transgene into the plant genome in each independently obtained transformant (position effect). It has been shown that in tobacco transformants expressing, for example, a cauliflower mosaic virus (CaMV) 35S promoter-driven -glucuronidase (GUS) reporter gene, these position-induced quantitative differences among individual transformants were reduced by the introduction of matrix-associated regions (MAR elements) on the T-DNA. We have previously shown by imaging of in planta firefly luciferase (luc) reporter gene activity that quantitative differences in transgene activity can be the result of either a variation in (1) level, (2) spatial distribution and/or (3) temporal regulation of transgene expression between independent transformants. It is not known which of these three different aspects of transgene expression is affected when the transgene is flanked by MAR elements. Here we have used the firefly luciferase reporter system to analyse the influence of MAR elements on the activity of a CaMV 35S-luc transgene in a population of independently transformed tobacco plants. Imaging of in planta LUC activity in these tobacco plant populations showed that the presence of MAR elements does not result in less variation in the average level of transgene expression between individual transformants. This result is different from that obtained previously with a 35S-GUS reporter gene flanked by MAR elements and reflects the differences in the stability of the LUC and GUS reporter proteins. Also the variation in spatial patterns of in vivo LUC activity is not reduced between independent transformants when the transgene is flanked by MAR elements. However, MAR elements do seem to affect the variation in temporal regulation of transgene expression between individual transformants. The potential effects of MAR elements on the variability of transgene expression and the relation to the stability of the (trans)gene product are discussed.     

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era. Streptomyces phage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through an attB/attP exchange, thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.     

Alternative promoters in expression of genetic information

Many genes are known to have several promoters. The contribution of alternative promoters to the structural and functional diversity of protein isoforms in eukaryotic cells is considered, including their role in synthesis of identical proteins from different mRNAs, generation of protein isoforms with different and even opposite functions, expression of housekeeping genes, and the variation of the recognition domains of adhesion proteins and receptors. In some cases, alternative promoters allow one gene to produce mRNAs with different open reading frames and, consequently, proteins with no amino acid sequence homology.     

Promoter regulation in Candida albicans and related species

Regulation of gene expression has been studied extensively in Saccharomyces cerevisiae and Schizosaccharomyces pombe . Some, but by far not all, of the findings are also applicable to Candida albicans , an important ascomycete fungal pathogen of humans. Areas of research in C. albicans include the influence of key signal transduction cascades on morphology, and the response to host-generated influences, such as host immune effector cells, blood, pH or elevated carbon dioxide. The resistance to antifungal agents and response to stress are also well researched. Conditional gene expression and reporter genes adapted to the codon usage of C. albicans are now widely used in C. albicans . Here we present a comprehensive overview of the current techniques used to investigate regulation mechanisms for promoters in C. albicans and other Candida species. In addition, we discuss reporter genes used for the study of gene expression.