Bicistronic expression of ecdysone-inducible receptors in mammalian cells

The recent emergence of inducible expression systems for mammalian cells has greatly facilitated the in vivo analysis of gene function. The ecdysone-inducible expression system is particularly attractive because of (i) extremely low basal expression and high-level induced expression, (ii) the lack of pleiotropic effects caused by the inducer or activator, and (iii) the rapid penetrance and clearance of the inducer. Here, we describe an improved receptor expression vector. The required ecdysone receptor proteins (VgEcR and RXR) are co-expressed from a bicistronic cytomegalovirus (CMV) expression cassette in the vector pERV3. The CMV promoter in this vector can be readily replaced with a cell type-specific promoter of interest. Using the ecdysone analogs, muristerone A or ponasterone A, induction ratios of up to three orders of magnitude were attained in the transient transfection assays and in a cell line stably transformed with both pERV3 and an ecdysone-inducible reporter vector. Fine control of luciferase expression was achieved bv varying both the induction time and inducer concentration. Here, we describe a set of cell lines stably transformed with the vector pERV3, in which the ecdysone receptors are expressed at optimal levels for the high-level induction of gene expression.     

Tetracycline-regulated gene expression switch in Xenopus laevis

Xenopus is a well-characterized model system for the investigation of biological processes at the molecular, cellular, and developmental level. The successful application of a rapid and reliable method for transgenic approaches in Xenopus has led to renewed interest in this system. We have explored the applicability of tetracycline-regulated gene expression, first described by Gossen and Bujard in 1992, to the Xenopus system. By optimizing conditions, tetracycline repressor induced expression of a luciferase reporter gene was readily and reproducibly achieved in both the Xenopus oocyte and developing embryo. This high level of expression was effectively abrogated by addition of low levels of tetracycline. The significance of this newly defined system for studies of chromatin dynamics and developmental processes is discussed.     

Manipulation of mitochondrial DNA gene expression in the mouse

Mitochondrial dysfunction due to impaired respiratory chain function is increasingly recognized as an important cause of human disease. Mitochondrial disorders are relatively common and have an estimated incidence of 1:10,000 live births. There are more than 100 different point mutations and numerous large rearrangements of mitochondrial DNA (mtDNA; mainly single deletions) that cause human disease. We aimed at obtaining an animal model to study physiological aspects of mtDNA mutation disorders. There are as yet unsolved technical problems associated with transfection of mammalian mitochondria. We therefore choose to manipulate mtDNA expression by targeting of the nuclear gene encoding Tfam. We utilised the cre-loxP recombination system to disrupt Tfam since this system allows manipulation of respiratory chain function in selected mouse tissues. We have found increased cell death or apoptosis induction in both germ line and tissue-specific Tfam knockouts. Our results further suggest that increased production of reactive oxygen species (ROS) is not a prominent feature in cells with impaired mtDNA expression.     

Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene

Strawberry (Fragaria x ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.     

FliZ induces a kinetic switch in flagellar gene expression

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.     

Manipulation of gene expression in zebrafish using caged circular morpholino oligomers

Morpholino oligomers (MOs) have been widely used to knock down specific genes in zebrafish, but their constitutive activities limit their experimental applications for studying a gene with multiple functions or within a gene network. We report herein a new design and synthesis of caged circular MOs (caged cMOs) with two ends linked by a photocleavable moiety. These caged cMOs were successfully used to photomodulate β-catenin-2 and no tail expression in zebrafish embryos.     

Effect of ecdysone receptor gene switch ligands on endogenous gene expression in 293 cells

Regulated gene expression may substantially enhance gene therapy. Correlated with structural differences between insect ecdysteroids and mammalian steroids, the ecdysteroids appear to have a benign pharmacology without adversely interfering with mammalian signaling systems. Consequently, the ecdysone receptor-based gene switches are attractive for application in medicine. In the present study, the effect of inducers of ecdysone receptor switches on the expression of endogenous genes in HEK 293 cells was determined. Four ligand chemotypes, represented by a tetrahydroquinoline (RG-120499), one amidoketone (RG-121150), two ecdysteroids [20-hydroxyecdysone (20E) and ponasterone A (Pon A)], and four diacylhydrazines (RG-102240, RG-102277, RG-102398 and RG-100864), were tested in HEK 293 cells. The cells were exposed to ligands at concentrations of 1 microm (RG-120499) or 10 microm (all others) for 72 h and the total RNA was isolated and analyzed using microarrays. Microarray data showed that the tetrahydroquinoline ligand, RG-120499 caused cell death at concentrations > or = 10 microm. At 1 microm, this ligand caused changes in the expression of genes such as TNF, MAF, Rab and Reprimo. At 10 microm, the amidoketone, RG-121150, induced changes in the expression of genes such as v-jun, FBJ and EGR, but was otherwise noninterfering. Of the two steroids tested, 20E did not affect gene expression, but Pon A caused some changes in the expression of endogenous genes. At lower concentrations pharmacologically relevant for gene therapy, intrinsic gene expression effects of ecdysteroids and amidoketones may actually be insignificant. A fortiori, even at 10 microm, the four diacylhydrazine ligands did not cause significant changes in expression of endogenous genes in 293 cells and therefore should have minimum pleiotropic effects when used as ligands for the ecdysone receptor gene switch.     

NF-kappaB-dependent regulation of tumor necrosis factor-alpha gene expression by CpG-oligodeoxynucleotides

Immunostimulatory activities of synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention as potentially useful immunotherapeutics. However, CpG-ODNs induce harmful and lethal shock effects because they greatly enhance the sequence-dependent induction of tumor necrosis factor-alpha (TNF-alpha). We have shown that phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) of the CpG-ODN 1826 stimulate TNF-alpha gene expression, TNF-alpha promoter activity, IkappaB degradation, and NF-kappaB activation at higher levels compared with its phosphodiester ODN (PO-ODN). In contrast to the effects of CpG-ODN 1826, PS-ODN of the CpG-ODN 2006 showed lower stimulatory activities than its PO-ODN. Using transient transfection, it was found that myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated factor 6 are commonly required for activation of the TNF-alpha promoter by various CpG-ODNs with different potencies. These results strongly suggest a possibility to optimally activate the innate immune responses by modulating the potency of CpG-ODNs via sequence rearrangement and phosphorothioate backbone modification.     

The oxygenation of murine tumor isografts and human tumor xenografts by nicotinamide.

The changes in pO2 caused by nicotinamide in the FSaII mouse tumor and three different xenografts of human tumors, HP-56, FaDu, and EO1, grown subcutaneously in the legs of mice were studied. The tumor pO2, as measured with microelectrodes, began to rise soon after the host mice were injected intraperitoneally with 500 mg/kg nicotinamide, and it increased continuously for 100-120 min. The rate and magnitude of the increase in tumor pO2 was dependent on the tumor line and also on the tumor size. In FSaII tumors, the increase in pO2 caused by nicotinamide was relatively small in the well-oxygenated small tumors (173 +/- 5 mm3) compared with that in the larger tumors (515 +/- 25 mm3). The blood perfusion in FSaII tumors as measured with the laser Doppler method was also increased by nicotinamide. The growth delay in FSaII tumors induced by X irradiation was enhanced significantly by nicotinamide. It was concluded that the enhancement of radiation damage in the experimental tumors in mice by nicotinamide, as observed in the present study and reported by others, is due to an increase in intratumor pO2, possibly as a result of an increase in blood perfusion.