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GFP Gene (gfp + gene)
Selected AbstractsLarge-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA libraryGENES TO CELLS, Issue 3 2000Da-Qiao Ding Background Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. Results We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5, end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products. Conclusions A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells. [source] Induction of the white egg 3 mutant phenotype by injection of the double-stranded RNA of the silkworm white geneINSECT MOLECULAR BIOLOGY, Issue 3 2002G. X. Quan Abstract Injection of double-stranded RNA (dsRNA) corresponding to the silkworm white gene (Bmwh3) into preblastoderm eggs of the wild-type silkworm induced phenotypes similar to those observed with mutants of the white egg 3 locus (10,19.6). The induced phenotypes were characterized by the presence of white eggs and translucent larval skin. Northern analysis showed that the expression of the endogenous Bmwh3 gene in the injected embryos was distinctly depressed. Furthermore, the injection of the GFP dsRNA inhibited the expression of the GFP gene from a plasmid co-injected with the dsRNA but did not depress the expression of the Bmwh3 gene. These findings demonstrate that sequence-specific RNA interference occurred in the silkworm. We conclude from the results that the RNA interference can be applied as a tool for the analysis of the gene function in the lepidopteran insects. [source] Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvaeINSECT MOLECULAR BIOLOGY, Issue 5 2001T. W. Ward Abstract Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [Afanasiev et al. (1994) Exp Parasitol 79: 322,339; Afanasiev et al. (1999) Virology 257: 62,72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139,159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations (Afanasiev et al., 1999; Allen-Muira et al. (1999) Virology 257: 54,61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day-old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10,0.15 m inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression. [source] Tenascin-C regulates angiogenesis in tumor through the regulation of vascular endothelial growth factor expressionINTERNATIONAL JOURNAL OF CANCER, Issue 1 2004Keiichiro Tanaka Abstract In order to verify whether tenascin-C (TN-C) is involved in angiogenesis as an extracellular signal molecule during tumorigenesis, cancerous cell transplantation experiments and coculture experiments were carried out, focusing on the regulation of vascular endothelial growth factor (VEGF). The A375 human melanoma cells introduced the GFP gene (A375-GFP), implanted subcutaneously into BALB/cA nude (WT) and TN-C knockout BALB/cA nude (TNKO) congenic mice. Furthermore, coculture experiments between A375-GFP and embryonic mesenchyme, which was prepared from both genotypes, were carried out to investigate the molecular mechanism in the cell-cell interactions. Both the content of TN-C and that of VEGF in the tumor and the conditioned medium were analyzed by the sandwich ELISA method. Seven days after transplantation of the A375-GFP, capillary nets became far more abundant in the tumors grown in WT mice than those in TNKO mice. Interestingly, VEGF and TN-C expressions showed antithetical expression patterns between the tumors in WT mice and those in TNKO mice. This peculiar phenomenon seems to be caused by a time lag prior to the onset of the mesenchymal regulation for the TN-C expression of A375-GFP. The coculture experiments revealed that WT mesenchyme had a much stronger effect than TNKO mesenchyme on both TN-C and VEGF expression. However, the defects of TNKO mesenchyme were restored in all cases by additional TN-C. These results clearly indicated that the expressions of both TN-C and VEGF depend on the surrounding mesenchyme, and that the function of mesenchyme is regulated by its own mesenchymal TN-C. In conclusion, the present data suggest that the matrix microenvironment organized by the host mesenchyme is very important for angiogenesis in tumor development. © 2003 Wiley-Liss, Inc. [source] Reduction of fibrosis in a rat model of non-alcoholic steatohepatitis cirrhosis by human HGF gene transfection using electroporationJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 8pt2 2008Shigeru Kiyama Abstract Background and Aim:, To study the histological changes caused by transfection of the hepatocyte growth factor (HGF) gene using electroporation (EP) in a non-alcoholic steatohepatitis (NASH) cirrhotic liver model. Methods:, NASH cirrhotic livers were prepared by administering a choline-deficient diet to 5-week-old male Wister rats for 12 weeks. Three groups of rats were used: rats in the G(+) group were transfected with the GFP gene using EP, rats in the H(+) group were transfected with the HGF gene using EP, and rats in the H(,) group were only injected with the HGF gene. Rats were sacrificed 2 days after gene transfection, and the Azan positive rate (APR) and Sudan positive rate (SPR) were calculated to evaluate fibrosis and fatty changes. Results:, The APR of the NASH cirrhotic livers was significantly higher than that in the normal livers. The APR did not decrease in the G(+) group and the H(,) group, but decreased significantly in the nonelectroporated as well as electroporated areas of the H(+) group. For SPR, there were no significant differences between the G(+), H(,), and H(+) groups. Conclusion:, The improvement of fibrosis was not significant when a direct injection of the HGF gene was used alone, but it was enhanced by the concomitant use of EP. However, no efficacy was observed in fat components. These findings suggest that transfection of the HGF gene by EP may lead to an improvement of irreversible cirrhotic livers to reversible fatty livers. [source] Gene transfer of disease regulated promoters during experimental autoimmune uveitisACTA OPHTHALMOLOGICA, Issue 2009V ELMALEH Purpose Adeno-associated virus (AAV) vectors have been successfully used to transfer immunosuppressive genes into the retina to prevent experimental uveitis development. Transgene expression is classically regulated by constitutive or tetracycline inducible promoters. It might be more advantageous that the control of transgene expression depends on the pathological process itself. Inflammation activates transcription factors acting on promoters containing short responsive sequences, responding, for example to nuclear factor kappa B (NF,B-RE). These responsive elements can be used to generate disease regulated promoters. Methods An AAV vector with the GFP gene under the control of a NF-kB-RE containing promoter will be injected subretinally in C57Bl6 mice. Autoimmune uveitis will be induced by adoptive transfer of IRBP specific lymphocytes. Animals will be sacrificed at different time points. GFP expression will be analysed by immunofluorescence. VCAM1, MHC II and CD45 will be analysed by immunofluorescence and used to monitor the level of retinal inflammation. Results One week after disease induction, GFP expression was found in eyes injected with this new vector. Milder GFP expression was also found in mice who did not received adoptive transfer. This background was increased a J14. Conclusion Our preliminary results suggest that disease driven GFP expression can be obtained by the use of AAV vectors containing disease regulated promoters. We still need some more times to improve our model. In the future, we plan to replace the GFP gene by an immunosuppressive gene and test if the system can be use to treat experimental uveitis. [source] HDAC inhibitor valproic acid enhances tumor cell kill in adenovirus-HSVtk mediated suicide gene therapy in HNSCC xenograft mouse modelINTERNATIONAL JOURNAL OF CANCER, Issue 3 2010Vishal Kothari Abstract Safety, efficacy and enhanced transgene expression are the primary concerns while using any vector for gene therapy. One of the widely used vectors in clinical trials is adenovirus which provides a safe way to deliver the therapeutic gene. However, adenovirus has poor transduction efficiency in vivo since most tumor cells express low coxsackie and adenovirus receptors. Similarly transgene expression remains low, possibly because of the chromatization of adenoviral genome upon infection in eukaryotic cells, an effect mediated by histone deacetylases (HDACs). Using a recombinant adenovirus (Ad-HSVtk) carrying the herpes simplex thymidine kinase (HSVtk) and GFP genes we demonstrate that HDAC inhibitor valproic acid can bring about an increase in CAR expression on host cells and thereby enhanced Ad-HSVtk infectivity. It also resulted in an increase in transgene (HSVtk and GFP) expression. This, in turn, resulted in increased cell kill of HNSCC cells, following ganciclovir treatment in vitro as well as in vivo in a xenograft nude mouse model. [source] A new molecular tool for transgenic diatomsFEBS JOURNAL, Issue 13 2005Control of mRNA, protein biosynthesis by an inducible promoter, terminator cassette Research in diatom biology has entered the postgenomic era since the recent completion of the Thalassiosira pseudonana genome project. However, the molecular tools available for genetic manipulation of diatoms are still sparse, impeding the functional analysis of diatom genes in vivo. Here we describe the first method for inducible gene expression in transgenic diatoms. This method uses a DNA cassette containing both promoter (Pnr) and terminator (Tnr) elements derived from the nitrate reductase gene of the diatom Cylindrotheca fusiformis. By using green fluorescent protein (gfp) cDNA as a reporter gene, it is demonstrated that gene expression under the control of the Pnr/Tnr cassette is switched off when cells are grown in the presence of ammonium ions and becomes switched on within 4 h when cells are transferred to medium containing nitrate. Incubating cells in nitrogen-free medium switches on transcription of the gfp gene, yet gfp mRNA does not become translated into protein. This block on translation is released by the addition of nitrate, resulting in rapid onset of GFP production with a drastically reduced delay time of only 1 h. Altogether we have demonstrated that the Pnr/Tnr cassette enables inducible gene expression and control of both the level and timing of mRNA and protein expression in transgenic diatoms. [source] The use of the green fluorescent protein as a biomarker for sapstain fungiFOREST PATHOLOGY, Issue 3 2002S. LEE To understand wood colonization by sapstain fungi and their potential biocontrol agents, it is necessary to differentiate these organisms directly on their natural substrates. In the present study the feasibility of transforming with the green fluorescent protein (GFP), the sapstain fungus Ophiostoma piceae and a potential biocontrol agent Cartapip®, an Ophiostoma piliferum albino strain was assessed. Transformants of the two fungal species were screened by polymerase chain reaction and Southern blot analyses. The GFP was expressed in spores, synnemata and mycelia of the transformants grown in artificial media or wood. The growth, pigmentation and wood colonization of the transformants were similar to that of the non-transformants, suggesting that the presence of the gfp gene had no negative effect on the biology of the transformants. Using fluorescence and confocal microscopy, the GFP-expressing fungi were easily differentiated from the wild-type strains and other fungal species in wood, even 4 months after inoculation. The results show that the use of the GFP system is feasible to monitor Ophiostoma fungi in wood. Utilisation de la protéine fluorescente verte (GFP) comme marqueur biologique des champignons de bleuissement du bois Pour comprendre la colonisation du bois par les champignons de bleuissement et par les agents de lutte biologique potentiels, il est nécessaire de distinguer ces organismes directement dans leur substrat naturel. Nous avons évalué la possibilité de transformation par la protéine fluorescente verte (GFP) du champignon de bleuissement Ophiostoma piceae et d'une souche albinos de Ophiostoma piliferum, agent de lutte biologique potentiel Cartapip®. Des transformants des deux espèces fongiques ont été triés par analyses PCR et Southern blot. La GFP a été exprimée dans les spores, les synnemas et le mycélium des transformants cultivés sur milieux artificiels et sur bois. Avec les transformants, la croissance, la pigmentation et la colonisation du bois étaient semblables à celles des non transformants, ce qui suggère que la présence du gène gfp n'a pas d'effet négatif sur la biologie des transformants. Par microscopie confocale à fluorescence, les champignons exprimant la GFP ont été facilement distingués des souches de type sauvage et d'autres espèces fongiques dans le bois, même 4 mois après inoculation. Nos résultats montrent que l'utilisation de la GFP est possible pour suivre les Ophiostoma dans le bois. Verwendung des Grünen Fluoreszenzproteins als Biomarker für Bläuepilze Um die Besiedelung von Holz durch Bläuepilze und ihre möglichen Antagonisten zu verstehen, muss man diese Organismen direkt auf ihrem natürlichen Substrat unterscheiden können. Es wurde überprüft, ob sich der Bläuepilze Ophiostoma piceae und der mögliche Antagonist Cartapip®, ein Albinostamm von Ophiostoma piliferum, mit dem Grünen Fluoreszenzprotein (GFP) transformieren lassen. Transformierte Stämme der beiden Pilzarten wurden mit PCR und Southern Blot Analysen untersucht. Das GFP wurde in Sporen, Synnemata und Myzelien der transformierten Stämme exprimiert. Dies war auf künstlichen Medien ebenso wie auf Holz der Fall. Wachstum, Pigmentierung und Holzbesiedelung waren bei den transformierten Stämmen ähnlich wie bei den nichttransformierten; somit dürfte die Präsenz des gfpGens keine negativen Auswirkungen auf die Biologie der transformierten Stämme haben. Mit Hilfe der Fluoreszenz- und Konfokal-Mikroskopie konnten die GFP exprimierenden Pilze leicht von den Wildtyp-Stämmen und anderen Pilzarten auf Holz unterschieden werden. Dies war auch noch vier Monate nach der Inokulation der Fall. Die Ergebnisse zeigen, dass das GFP-System zur Beobachtung von Ophiostoma -Arten im Holz geeignet ist. [source] Viroid-induced RNA silencing of GFP-viroid fusion transgenes does not induce extensive spreading of methylation or transitive silencingTHE PLANT JOURNAL, Issue 1 2004Ulrike Vogt Summary Viroid infection is associated with the production of short interfering RNAs (siRNAs), a hallmark of post-transcriptional gene silencing (PTGS). However, viroid RNAs autonomously replicating in the nucleus have not been shown to trigger the degradation of homologous RNA in the cytoplasm. To investigate the potential of viroids for the induction of gene silencing, non-infectious fragments of potato spindle tuber viroid (PSTVd) cDNA were transcriptionally fused to the 3, end of the green fluorescent protein (GFP)-coding region. Introduction of such constructs into tobacco plants resulted in stable transgene expression. Upon PSTVd infection, transgene expression was suppressed and partial de novo methylation of the transgene was observed. PSTVd-specific siRNA was detected but none was found corresponding to the gfp gene. Methylation was restricted almost entirely to the PSTVd-specific part of the transgene. Neither a gfp transgene construct lacking viroid-specific elements was silenced nor was de novo methylation detected, when it was introduced into the genetic background of the PSTVd-infected plant lines containing silenced GFP:PSTVd transgenes. The absence of gfp -specific siRNAs and of significant methylation within the gfp -coding region demonstrated that neither silencing nor DNA methylation spread from the initiator region into adjacent 5, regions. [source] | |||||||||||||