Inducible expression of Norwalk virus capsid protein gene in plant cell suspension cultures

Plant cell suspension cultures can be used to make safe vaccines at a lower cost than conventional procedures. An inducible gene expression system provides an opportunity to optimize the conditions of vaccine production in a plant system. In this investigation, a dexamethasone-inducible Norwalk virus capsid protein (NVCP) gene expression system has been developed in cell suspension cultures for four different plant species: tobacco (Nicotiana tabacum), rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and slash pine (Pinus elliottii Engelm.) via Agrobacterium-mediated transformation. Resulting transgenic cell lines were confirmed by Southern blot analyses and NVCP gene expression was confirmed by Northern blot analysis. NVCP gene expression was observed in all 24 cell lines tested, but there were minor differences in transgene expression among the transgenic cell lines. The highest level of NVCP gene expression was observed 48 h after addition of the glucocorticoid hormone dexamethasone (10 mg/l), for all transgenic cell lines derived from four different plant species. This investigation demonstrated that expression of NVCP in different transgenic cell lines and in different species was tightly controlled by the inducer, and the inducible gene expression system could be useful in controlling expression of NVCP or similar proteins for production of vaccines in cultured plant cells.     

Characterization of the yeast copper-inducible promoter system in Arabidopsis thaliana

Inducible promoters or gene-switches are used to both spatially and temporally regulate gene expression. Such regulation can provide information concerning the function of a gene in a developmental context as well as avoid potential harmful effects due to overexpression. A gfp construct under the control of a copper-inducible promoter was introduced into Arabidopsis thaliana (L.) Heynh. and the regulatory parameters of this inducible promoter were determined. Here, we describe the time-course of up- and down-regulation of GFP expression in response to copper level, the optimal regulatory levels of copper, and the tissue specificity of expression in three transgenic lines. We conclude that the copper-inducible promoter system may be useful in regulating the time and location of gene expression in A. thaliana.     

High efficiency inducible gene expression system based on activation of a chimeric transcription factor in transgenic pine

Inducible gene expression systems are needed in functional genomics of tree species. A glucocorticoid-inducible gene expression system was established in a gymnosperm species Virginia pine (Pinus virginiana Mill.) through Agrobacterium tumefaciens-mediated genetic transformation. The results demonstrate that expression of the m-gfp5-ER reporter gene was tightly controlled and 0.1 microM of the glucocorticoid hormone triamcinolone was able to induce m-gfp5-ER expression in transgenic cells. Differential expression of gfp in transgenic cells induced by different concentrations of triamcinolone was observed and confirmed by Northern Blot analysis and by quantitative green fluorescence analyses with Laser Scanning Microscopy. In transgenic plantlets, triamcinolone was taken up efficiently by roots. Triamcinolone was able to induce m-gfp5-ER activity throughout the whole plant. The phenotype of transgenic plantlets was not affected 6 weeks after treatment with 0.1-10 microM triamcinolone. However, 6-week inductions with 100 microM triamcinolone caused growth retardation and developmental defects, as well as inhibition of root formation and elongation. With careful selection of transgenic lines, the inducible gene expression presented in this study could be a very valuable alternative for functional identification of novel genes in plants, especially in pine.     

Transgenic Christmas Trees Regenerated from Agrobacterium tumefaciens-mediated Transformation of Zygotic Embryos Using the Green Fluorescence Protein as a Reporter

Mature zygotic embryos of recalcitrant Christmas tree species Fraser fir [Abies fraseri (Pursh) Poir], and Nordmann fir (Abies nordmanniana L.k.), and Virginia pine (Pinus virginiana Mill.) were used as explants for Agrobacterium tumefaciens strain GV3850-mediated transformation using the gfp (green fluorescent protein) gene as a reporter. Factors including media used for inoculation and co-cultivation, concentrations of acetosyringone, and antibiotics in tissue culture media have been evaluated. A high transformation frequency was obtained on TE medium containing 50μM acetosyringone and using 500 mg/l timentin to eliminate bacteria. Transient gene expression was observed in all three Christmas tree species, but transgenic plants were only produced from Virginia pine. Stable integration and expression of transgenes in the plant genome of Virginia pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable transformation system has been established in Virginia pine and this system would provide an opportunity to transfer economically important genes into Christmas tree species.     

染色体重组对转基因大麦中gfp基因表达的作用

利用5种转绿色荧光蛋白基因(gfp)大麦不同株系及野生型大麦为材料,对不同转基因株系间以及转基因株系与野生型植株间进行杂交,分别对不同世代植株的根尖、花粉中gfp基因的表达量进行测定.结果表明,不同转基因株系间的根尖、花粉的gfp基因在表达量上存在差异,同一转基因材料的gfp基因表达存在组织差异;gfp基因在杂交后代中作为一个显性基因以孟德尔方式稳定遗传,不同染色体上的gfp基因重组有利于提高持基因表达.     

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.     

A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action

Cre-mediated site-specific recombination allows conditional transgene expression or gene knockouts in mice. Inducible Cre recombination systems have been developed to bypass initial embryonic lethal phenotypes and provide access to later embryonic or adult phenotypes. We have produced Cre transgenic mice in which excision is tamoxifen inducible and occurs in a widespread mosaic pattern. We utilized our Cre excision reporter system combined with an embryonic stem (ES) cell screen to identify ES cell clones with undetectable background Cre activity in the absence of tamoxifen but efficient excision upon addition of tamoxifen. The CreER transgenic mouse lines derived from the ES cells were tested using the Z/AP and Z/EG Cre reporter lines. Reporter gene expression indicated Cre excision was maximal in midgestation embryos by 2 days after tamoxifen administration, with an overall efficiency of 5-10% of cells with Cre excision. At 3 days after tamoxifen treatment most reporter gene expression marked groups of cells, suggesting an expansion of cells with Cre excision, and the proportion of cells with Cre excision was maintained. In adults, Cre excision was also observed with varying efficiencies in all tissues after tamoxifen treatment.     

Expression of the E3L gene of vaccinia virus in transgenic mice decreases host resistance to vaccinia virus and Leishmania major infections

The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2′-5′-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations.     

Tet system的调控原理及其在转基因小鼠模型上的应用

基因表达的调控是分子生物学研究的一个重要问题,也是基因治疗和基因功能研究的重要手段。诱导性基因表达系统可以从时间上调控基因的表达,是基因治疗和基因功能研究的重要工具之一。其中,四环素诱导基因表达系统(tetracycline inducible expression system,Tet system)是应用最广泛的一种,它可以在时间和空间上对基因进行严谨和高效地诱导表达。基于该系统获得了不同用途的转基因动物,这些模型动物的建立为研究特定基因的功能及其在疾病发生中的作用打下了实验基础。现就四环素诱导表达系统的原理和在小鼠模型上的研究应用做一综述。     

重组DNA技术中可诱导的基因表达调控系统

在重组DNA技术中,可诱导的基因表达调控系统可被用来调节目的基因的表达以达到基因功能研究、转基因动物研究、以及基因治疗研究等目的。该系统主要由诱导剂、可诱导的受体或转录因子、顺式作用元件以及载体系统四部分组成。本文以诱导剂为分类依据,叙述目前主要的6类可诱导的基因表达调控系统:类固醇激素受体诱导的基因表达调控系统、四环素诱导的基因表达调控系统、缺氧诱导的基因表达调控系统、高热诱导的基因表达调控系统、电离辐射诱导的基因表达调控系统和lac基因表达调控系统。     

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.