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Results for “Transient expression”

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PubMed2005

Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants.

Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants: stable genetic transformation and transient expression using viral vectors. Although much faster, the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes. We have developed constructs for the efficient delivery of RNA viral vectors as DNA precursors and show here that Agrobacterium-mediated delivery of these constructs results in gene amplification in all mature leaves of a plant simultaneously (systemic transfection). This process, called "magnifection", can be performed on a large scale and with different plant species. This technology combines advantages of three biological systems (the transfection efficiency of A. tumefaciens, the high expression yield obtained with viral vectors, and the post-translational capabilities of a plant), does not require genetic modification of plants and is faster than other existing methods.

Agrobacterium tumefaciens↗

PubMed1999

Large-scale transient expression in mammalian cells for recombinant protein production.

Large-scale transient expression from mammalian cells is a new technology. Breakthroughs have been achieved for non-viral delivery methods: transfections can now be done at the 1-10 L scale with mammalian cells grown in suspension. Production of 1-20 mg/L of recombinant protein have been obtained in stirred bioreactors. Modified alphaviruses have provided a fast and efficient expression technology based on viral vectors.

Adenoviridae↗

PubMed2002

Characterization of cloned mouse Na+/taurocholate cotransporting polypeptide by transient expression in COS-7 cells.

The mouse Na+/taurocholate cotransporting polypeptide transiently expressed in COS-7 cells caused sodium-dependent uptake of [3H]taurocholic acid with Km and Vmax values of 18 microM and 102 pmol/mg protein/min, respectively. This Km value is comparable to that for rat NTCP and higher than that for human NTCP. Substrate specificity was evaluated by measuring inhibitory effects of unlabeled bile acids on [3H]taurocholic acid transport.

Animals↗

PubMed2003

Transient expression of homologous hairpin RNA causes interference with plant virus infection and is overcome by a virus encoded suppressor of gene silencing.

Specific post-transcriptional gene silencing (PTGS) of target genes can be induced in a variety of organisms by providing homologous double-stranded RNA (dsRNA) molecules. In plants, PTGS is part of a defense mechanism against virus infection. We have previously shown and patented that direct delivery to nontransgenic plants of dsRNA derived from viral sequences specifically interfere with virus infection. Here, we show that transient expression of constructs encoding hairpin RNA homologous to a rapidly replicating plant tobamovirus also interferes with virus multiplication in a sequence-dependent manner. A three-day lag period between delivery of hairpin RNA and virus into the same tissues completely block virus infectivity. Several hallmarks characteristic of PTGS were associated with viral interference mediated by hairpin RNA: high level of sequence identity between the hairpin RNA and the target RNA, presence of siRNAs in extracts derived from leaves infiltrated with hairpin RNA, and helper component-proteinase (HC-Pro) of potyviruses, a suppressor of PTGS, overcame interference. No evidence for a mobile silencing suppression signal induced by transient expression of HC-Pro was observed. The approach described here has the potential to be used as a versatile tool for studying the onset of PTGS in cases involving virus infection, in opposition to dsRNA-transgenic plants, which allow primarily for the study of PTGS maintenance.

Gene Silencing↗

PubMed1993

A phosphorylation epitope on MAP 1B that is transiently expressed in growing axons in the developing rat nervous system.

We have isolated a monoclonal antibody (150) that recognizes a phosphorylation epitope on the microtubule-associated protein (MAP) 1B. Immunoblot analysis of the developing rat central nervous system shows that monoclonal antibody 150 is directed against a protein of approximately 325 kDa (MAP 1B) that copolymerizes with microtubules through successive cycles of temperature-dependent assembly and disassembly. Furthermore, immunoprecipitated MAP 1B contains the epitope recognized by monoclonal antibody 150. Removal of phosphate from blotted proteins using alkaline phosphatase abolishes the binding of monoclonal antibody 150 to MAP 1B, indicating that the epitope is phosphorylated. In the developing rat nervous system, immunohistochemistry with monoclonal antibody 150 shows that the phosphorylation epitope on MAP 1B is transiently expressed in growing axons but not in dendrites. For instance, in the neonatal rat cerebellum, the parallel fibres of granule cells are stained only during elongation and not after synaptogenesis. The monoclonal antibody 150 epitope is also transiently expressed in radial glial fibres and in certain cell nuclei. All immunostaining of sections with monoclonal antibody 150 was completely abolished by alkaline phosphatase treatment. These observations and previous ones made by us in cell culture (Mansfield et al., J. Neurocytol., 20, 654-666, 1991) suggest that the phosphorylation epitope on MAP 1B recognized by monoclonal antibody 150, which has not been previously detected in vivo, may be important in axonogenesis.

Aging↗

PubMed1997

Calretinin is transiently expressed in tendon fibroblasts of the paravertebral region of the chick embryo.

Although calretinin is an intracellular calcium acceptor protein essentially located in neurons, we have previously shown that calretinin was also expressed in the mesenchymal cells forming the intervertebral disc. Here, we describe, using immunohistochemistry, the transient expression of calretinin in fibroblasts responsible for the tendon formation in the paravertebral region. We have looked at chick embryos from embryonic day 4 to day 18. At embryonic day 6, calretinin immunoreactivity was detected in chick embryo cells located laterally to the spinal cord between two groups of developing muscular cells. At embryonic day 8, calretinin immunoreactivity intensity was the highest in cells showing a fibroblast morphology. After embryonic day 8, when fibroblast proliferation decreased and differentiation increased, calretinin immunoreactivity progressively disappeared. Interestingly, calretinin could not be detected in fibroblasts of the anterior and posterior limbs at any investigated age. Because calretinin expression appeared selectively and transiently in the fibroblasts of the paravertebral region, we may conclude that the phenotype of those fibroblasts is different from the limbs fibroblasts.

Animals↗

PubMed1999

Transient expression of members of the germin-like gene family in epidermal cells of wheat confers disease resistance.

The wheat genome encodes a family of germin-like proteins that differ with respect to regulation and tissue specificity of expression of the corresponding genes. While germin exhibits oxalate oxidase (E.C. 1.2.3.4.) activity, the germin-like proteins (GLPs) have no known enzymatic activity. A role of oxalate oxidase in plant defence has been proposed, based on the capacity of the enzyme to produce H2O2, a reactive oxygen species. The role in defence of germin and other members of the germin-like gene family was functionally assessed in a transient assay system based on particle bombardment of wheat leaves. Transient expression of the pathogen-induced germin gf-2.8 gene, but not of the constitutively expressed HvGLP1 gene, reduced the penetration efficiency of Blumeria (syn. Erysiphe) graminis f.sp. tritici, the causal agent of wheat powdery mildew, on transformed cells. Two engineered germin-gf-2.8 genes and the TaGLP2a gene, which all encoded proteins without oxalate oxidase activity, also reduced the penetration efficiency of the fungus, demonstrating that oxalate oxidase activity is not required for conferring enhanced resistance. Instead, activity tagging experiments showed that in cells transiently expressing the germin gf-2.8 gene, the transgene product became insolubilized at sites of attempted fungal penetration where localised production of H2O2 was observed. Thus, germin and GLPs may play a structural role in cell-wall re-enforcement during pathogen attack.

Base Sequence↗

PubMed1995

Transient expression of S-100 by melanotropes of the rat pituitary intermediate lobe during development.

S-100, an acidic calcium-binding protein, is co-localized with vimentin in glial-like cells in the adult rat pituitary intermediate lobe. S-100 and melanotrope markers were not co-localized in the adult. During development, S-100 and vimentin were not co-localized but appeared in cells with different morphological characteristics. S-100 was co-localized with POMC mRNA and beta-endorphin during prenatal time and the first three postnatal weeks. This was demonstrated by double-label immunohistochemistry, using combinations of antisera against S-100, vimentin and beta-endorphin, and in situ hybridization histochemistry for POMC mRNA combined with immunohistochemistry for S-100. In the second and third weeks of postnatal development, S-100 was observed in fewer melanotropes and more frequently in stellate cells, which also expressed vimentin. Thus, S-100 appeared to be transiently expressed in melanotropes during prenatal and early postnatal development. S-100 serves as a neurotrophic and glial maturation factor in the CNS. Since S-100 expression in melanotropes coincides with the onset of dopaminergic innervation and morphological changes in glial-like cells of the lobe, it could have similar functions in the rat pituitary intermediate lobe.

Animals↗

PubMed2000

Heparan N-sulfatase gene: two novel mutations and transient expression of 15 defects.

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) results from the deficiency of the enzyme heparan N-sulfatase (NS, EC 3.10.1.1), required for the degradation of heparan sulfate. Molecular defects of 24 Italian MPS IIIA patients were recently reported by our group. We report here two novel mutations: 1040insT and Q365X and the expression studies on 15 of the identified defects. Transient expression of COS cells by cDNA mutagenized to correspond to heparan N-sulfatase mutations Y40N, A44T, 166delG, G122R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E369K, R377C did not yield active enzyme, demonstrating the deleterious nature of the mutations. Western blot analysis and metabolic labeling experiments revealed, for cells transfected with wild-type enzyme, a precursor 62-kDa form and a mature 56-kDa form. Western blot resulted, for 11 mutations, in the presence of both forms, indicating a normal maturation of the mutant enzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increased degradation of the mutant enzymes.

Animals↗

PubMed1995

Collagen gene expression during development of avian synovial joints: transient expression of types II and XI collagen genes in the joint capsule.

The developmental sequence of the embryonic joint has been well studied morphologically. There are, however, no definitive studies of cell function during joint development. In order to begin to understand the differentiation events that contribute to joint formation, we examined the expression of collagen mRNAs encoding types I, IIA, IIB, and XI. In situ hybridization was performed on chicken embryo hind limb buds and digits from day 7 to day 18 (Hamburger and Hamilton stages 31-44). In the day 7 (stage 31) limb bud, there was a condensation of mesenchyme forming the primitive tarsal and metatarsal bones that showed abundant expression of type IIA procollagen message, but no type IIB or type alpha 1(XI) message. By day 8 (stage 33), co-expression of types IIA, and type XI procollagen mRNAs was observed in the condensations, with expression of IIB restricted to early chondrocytes with metachromatically staining matrix. At this stage, DNA fragmentation characteristic of apoptosis was observed in cells near the midline of the interzone region between the developing anlagen, and in areas between and around the individual digits of the paddle. The presumptive apoptotic cells were more numerous at day 9 (stage 35), and were not found in the developing joint at subsequent time points, including the initiation of spatial cavitation of the joint. From days 11-18, type IIA procollagen mRNA was expressed in flattened cells at the surface of the anlagen, and in the perichondrium and in the developing joint capsule; type IIB mRNA message was found only in chondrocytes. Type XI mRNA was expressed by all type II-expressing cells. Alpha 1(I) mRNA was expressed early by cells of the interzone and capsule, but as cavitation progressed, the type I expressing cells of the interzone merged with the superficial layer of the articular surface. Thus, at the time of joint cavitation, there was a distinct pattern of expression of procollagen messages at the articular surface, with type I being outermost, followed by morphologically similar cells expressing type IIA, then chondrocytes expressing type IIB. The progenitor cells expressing type IIA message define a new population of cells. These cell populations contribute to the molecular heterogeneity of the articular cartilage, and these same populations likely exist in the developing joints of other species. The transient transcription of type II and type XI collagen genes, characteristic of chondrocytes, by cells in the joint capsule demonstrates that these cells may have chondrogenic potential.

Animals↗

PubMed1999

Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene.

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T1 progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.

Agrobacterium tumefaciens↗

PubMedDate not supplied

Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells.

BACKGROUND: Tumor vaccines, which are created by the insertion of cDNA encoding different cytokines into the tumor cells, are capable of inducing a very complex immune reaction including activation of CD8 T cells, granulocytes, macrophages, the triggering of cytokine cascades and antibody production. Aiming to create genetically modified tumor cells which could produce and secrete Human Tumor Necrosis Factor-alpha (hTNF-alpha), we constructed the expression cassette containing hTNF-alpha gene in pcDNA3 plasmid vector. MATERIALS AND METHODS: The successful ligation of cDNA encoding for hTNF-alpha into pcDNA3 plasmid vector was confirmed by PCR, restriction mapping and sequence determination. The constructed expression cassette in pcDNA3 vector was than transferred in vitro into malignant melanoma B16 tumor cells by the method of Receptor Mediated Gene Transfer (RMGT). RESULTS: Measurable amounts of hTNF-alpha protein detected in the medium of transfected cells proved that tumor cells modified in this manner became producers of hTNF-alpha protein. CONCLUSION: The expression of the transferred gene was transient and the produced protein was biologically active. Furthermore, the production of hTNF-alpha protein was also observed in sub-lethally irradiated tumor cells, showing that the expression cassette was preserved during the irradiation and that the cells were potentially applicable as a tumor vaccine.

Animals↗

PubMed1992

Transient expression of adheron molecules during chick retinal development.

Neuritogenesis and synapse formation are transient phenomena mediated in part by filopodial attachments (Tsui, Lankford, and Klein, Proc. Natl. Acad. Sci. 82:8256-8260 1985). The attachments can be labeled by antisera against adherons, adhesive microparticles isolated from cell culture media (Tsui, Schubert, and Klein, J. Cell Biol. 106:2095-2108 1988). Here, two monoclonal antibodies raised against adherons have been found to recognize transiently expressed membrane antigens of developing avian retina. Early in development, monoclonal antibody (mAb) AD1 stained antigens that spanned the entire tissue. With time, immunoreactivity became restricted to optic fiber, ganglion cell, and inner plexiform layers. Immunoblots of embryonic day (E) 13 retina showed a broad band at 66-72 kD for particulate fractions and a fine band at 70 kD for soluble fractions. The particulate forms disappeared as retinas matured, but the soluble form did not. mAb AD2 initially labeled retina antigens of optic fiber, ganglion cell, and inner plexiform layers (IPL). Labeling in the plexiform layer showed discrete lamina. Immunoreactivity first appeared at E9, peaked at E15, and then disappeared shortly after hatching. In isolated cells, AD2 labeled small cell surface aggregates. Cytoarchitectural studies, using whole-mount transmission electron microscopy, showed AD2 antigen in cell surface microfilaments, including some that joined filopodia together. The adheron antigens recognized by mAbs AD1 and AD2 thus were (1) topographically restricted; (2) associated with cell surfaces; and (3) developmentally down-regulated. This pattern suggests a role in developmentally transient cell surface phenomena, such as neurite extension or junction biogenesis.

Animals↗

PubMed1997

Cadherin-6 expression transiently delineates specific rhombomeres, other neural tube subdivisions, and neural crest subpopulations in mouse embryos.

Mammalian cadherin-6 (K-cadherin, cad6) was originally identified by means of the polymerase chain reaction, but its biological functions have not yet been determined. We analyzed the expression pattern of the mouse homologue of this cadherin during development and found that it was transiently expressed in restricted rhombomeres and in other subdivisions of the neural plate and tube. In the midbrain and anterior hindbrain of E8.0-8.5 embryos, cad6 was expressed only in neural crest-generating regions. In contrast, in the posterior hindbrain and contiguous spinal cord of these embryos, cad6 occurred throughout the neural plate, forming a sharp anterior limit at the future rhombomere 4 and 5 boundary. Subsequently, this neural plate expression became confined to rhombomere 6, although most of the neural crest-generating areas remained positive throughout the body. Neural crest cells expressing cad6 migrated out of the neural tube, and subsequently accumulated mainly along peripheral nerves. We then studied the effect of Hoxa-1 mutation on the expression of cad6, as their expressions spatiotemporally overlapped with each other in the early posterior hindbrain. In E8.0-8.5 Hoxa-1 mutants, cad6 expression was suppressed in the region of rhombomeres 4 to 6, although that in the other regions was not essentially affected. At later stages, however, cad6-positive crest cells appeared and migrated out of rhombomeres 4 to 6, indicating that the suppression of cad6 expression was transient and restricted to early stages. Importantly, this effect of the Hoxa-1 mutation concurred with the timing of the expression of this gene. We also studied Hoxa-3 mutants, but found no effect of this mutation on the cad6 expression pattern. These findings suggest that cad6 may contribute to the formation of the segmental structure of the early brain through its ability to confer specific adhesiveness on cells and that Hoxa-1 may be required for early cad6 expression in the posterior hindbrain.

Amino Acid Sequence↗

PubMed1997

Long-term expression of Fos-related antigen and transient expression of delta FosB associated with seizures in the rat hippocampus and striatum.

Systemic administration of kainic acid (KA), an analogue of glutamic acid, causes limbic seizures and pathophysiological changes in adult rats that are very similar to human temporal lobe epilepsy. One of the earliest changes in gene expression after treatment with KA is the induction of immediate-early genes. The fos and jun families are frequently studied immediate-early genes that are induced by KA. Several groups, including ours, have recently reported that a 35-kDa Fos-related antigen (FRA) is induced for a protracted time by various stimuli. It has been suggested that this FRA is delta FosB, which has a molecular mass of approximately 35 kDa. The present study characterizes the long-term expression of FRA and delta FosB after systemic treatment with KA. Immunocytochemistry and western blot analysis using an antibody that cross-reacts with all known FRAs showed that a 35-kDa FRA was induced at high levels in both the hippocampus and striatum for up to 1 month by KA. A semiquantitative PCR analysis showed that delta FosB was induced by KA, but its expression lasted for only 6 h. This result was also verified by northern blot analysis. These results suggested that the 35-kDa FRA with long-term elevated levels seen with western blot analysis and immunocytochemistry is a new species of the FRA and not delta FosB. The long-term expression of FRA in both the hippocampus and striatum may be associated with the pathophysiological changes after KA administration.

Animals↗

PubMed1992

Transient expression assay in a baculovirus system using firefly luciferase gene as a reporter.

Transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) in insect cells of Bombyx mori and Spodoptera frugiperda, respectively. DNA sequences encoding luciferase (luc) of the firefly Photinus pyralis was successfully employed in the expression assay as a reporter gene. Recombinant plasmids were constructed containing the luc gene under control of baculovirus-specific or heterologous promoters. Cotransfection of Bombyx mori and Spodoptera frugiperda cells with recombinant plasmids carrying virus-specific promoter sequences and BmNPV and AcNPV DNA, respectively, gave rise to efficient synthesis of luciferase (Luc), while heterologous promoters induced a low level of luc expression. We found that flanking sequences of the AcNPV DNA in the transfer plasmid contained an unknown promoter conferring an efficient luc expression. The activity of this promoter was modulated by the polh promoter sequences. The assay allows one to conduct highly sensitive monitoring of the transient expression of foreign genes from the transfecting plasmids prior to construction of recombinant viruses.

Animals↗

PubMed2006

A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean.

Fluorescent proteins such as green fluorescent protein (GFP) from Aequorea victoria are often used as markers for transient expression and stable transformation in plants, given that their detection does not require a substrate and they can be monitored in a nondestructive manner. We have now evaluated the red fluorescent protein DsRed2 (a mutant form of DsRed from Discosoma sp.) for its suitability as a visual marker in combination with antibiotic selection for genetic transformation of soybean [Glycine max (L.) Merrill]. Transient and stable expression of DsRed2 in somatic embryos was readily detected by fluorescence microscopy, allowing easy confirmation of gene introduction. We obtained several fertile transgenic lines, including homozygous lines, that grew and produced seeds in an apparently normal manner. The red fluorescence of DsRed2 was detected by fluorescence microscopy without background fluorescence in both leaves and seeds of the transgenic plants. Furthermore, in contrast to seeds expressing GFP, those expressing DsRed2 were readily identifiable even under white light by the color conferred by the transgene product. The protein composition of seeds was not affected by the introduction of DsRed2, with the exception of the accumulation of DsRed2 itself, which was detectable as an additional band on electrophoresis. These results indicate that DsRed2 is a suitable reporter (even more suitable than GFP) for genetic transformation of soybean.

Animals↗

PubMed1996

High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos.

For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.

Actins↗