The Experts below are selected from a list of 19470 Experts worldwide ranked by ideXlab platform
Jon A Wolff - One of the best experts on this subject based on the ideXlab platform.
-
intraarterial delivery of naked plasmid dna expressing full length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy
Human Gene Therapy, 2004Co-Authors: Goufeng Zhang, Hans Herweijer, James J Ludtke, Christine Thioudellet, Patricia Kleinpeter, Michael Antoniou, Serge Braun, Jon A WolffAbstract:Our previous studies have demonstrated that the intraarterial delivery of naked plasmid DNA leads to high levels of Foreign Gene expression throughout the muscles of the targeted limb. Although the procedure was first developed in rats and then extended to nonhuman primates, the present study has successfully implemented the procedure in normal mice and the mdx mouse model for Duchenne muscular dystrophy. After intraarterial delivery of plasmid DNA expressing the normal, full-length mouse dystrophin from either the cytomegalovirus promoter or a muscle-specific human desmin Gene control region, mdx mouse muscle stably expressed dystrophin in 1-5% of the myofibers of the injected hind limb for at least 6 months. This expression Generated an antibody response but no apparent cellular response.
-
efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates
Human Gene Therapy, 2001Co-Authors: Guofeng Zhang, Vladimir G Budker, Phillip Williams, Vladimir M Subbotin, Jon A WolffAbstract:We have previously shown that the intraarterial delivery of naked plasmid DNA (pDNA) into the femoral artery of rats leads to high levels of Foreign Gene expression throughout the muscles of the hindlimb. The present study shows that the procedure can also enable high levels of Foreign Gene expression throughout the limb skeletal muscles in rhesus monkeys. The average luciferase expression in the target muscle was 991.5 ± 187 ng/g for the arm and 1692 ± 768 ng/g for the leg; compared with 780 ng/g in rat hindlimb. Large numbers of β-galactosidase-positive myofibers were found in both leg and arm muscles, ranging from less than 1% to more than 30% in various muscles, with an average of 6.9%. The nonhuman primates tolerated the procedure without significant adverse effects in skeletal muscles, arteries, or other organs. Other studies in immunosuppressed rats indicated that stable expression is possible. These results suggest that the procedure is likely to enable efficient and stable Gene expression in huma...
-
high levels of Foreign Gene expression in hepatocytes after tail vein injections of naked plasmid dna
Human Gene Therapy, 1999Co-Authors: Guofeng Zhang, Vladimir G Budker, Jon A WolffAbstract:We have previously shown that the intramuscular injection of naked plasmid DNA enables Foreign Gene expression in muscle. Further studies showed that the intravascular delivery of naked plasmid DNA enables high levels of expression not only in muscle but also in hepatocytes. For the liver, this technique required injection directly into the liver vessels (portal vein, hepatic vein, or bile duct) and occlusion of outflow. The present study now demonstrates that high levels of plasmid DNA expression in hepatocytes can be easily obtained by tail vein injections. The highest levels of expression are achieved by rapidly injecting the plasmid DNA in large volumes, ~2.5 ml. This technique has great potential for a wide variety of laboratory studies.
-
high expression of naked plasmid dna in muscles of young rodents
Human Molecular Genetics, 1997Co-Authors: Istvan Danko, Guofeng Zhang, Phillip Williams, Hans Herweijer, Jeffrey S Latendresse, Ildiko Bock, Jon A WolffAbstract:There is a time window at 2 weeks of age for achieving very high levels of Foreign Gene expression from the intramuscular injection of naked plasmid DNA in mice and rats. The highest expression, over 1 microg of luciferase protein/muscle, was obtained in Balb/C mice using constructs containing the CMV promoter, a chimeric intron and the luc+ luciferase Gene. Approximately 50% of the myofibers were intensely blue following the intramuscular injection of a beta-galactosidase expression vector in 2 week old Balb/C mice. The effects of age, mouse strain and construct were multiplicative, resulting in >1000-fold greater luciferase and approximately 20-fold more beta-galactosidase-positive cells. These high levels of expression were unstable and were not observed in larger animals (dog, rhesus monkey). These results indicate that enormous levels of Foreign Gene expression can be obtained in muscle with naked DNA in vivo and will enable the temporary effects of Gene function and expression in rodent muscle to be expeditiously studied.
Guofeng Zhang - One of the best experts on this subject based on the ideXlab platform.
-
efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates
Human Gene Therapy, 2001Co-Authors: Guofeng Zhang, Vladimir G Budker, Phillip Williams, Vladimir M Subbotin, Jon A WolffAbstract:We have previously shown that the intraarterial delivery of naked plasmid DNA (pDNA) into the femoral artery of rats leads to high levels of Foreign Gene expression throughout the muscles of the hindlimb. The present study shows that the procedure can also enable high levels of Foreign Gene expression throughout the limb skeletal muscles in rhesus monkeys. The average luciferase expression in the target muscle was 991.5 ± 187 ng/g for the arm and 1692 ± 768 ng/g for the leg; compared with 780 ng/g in rat hindlimb. Large numbers of β-galactosidase-positive myofibers were found in both leg and arm muscles, ranging from less than 1% to more than 30% in various muscles, with an average of 6.9%. The nonhuman primates tolerated the procedure without significant adverse effects in skeletal muscles, arteries, or other organs. Other studies in immunosuppressed rats indicated that stable expression is possible. These results suggest that the procedure is likely to enable efficient and stable Gene expression in huma...
-
high levels of Foreign Gene expression in hepatocytes after tail vein injections of naked plasmid dna
Human Gene Therapy, 1999Co-Authors: Guofeng Zhang, Vladimir G Budker, Jon A WolffAbstract:We have previously shown that the intramuscular injection of naked plasmid DNA enables Foreign Gene expression in muscle. Further studies showed that the intravascular delivery of naked plasmid DNA enables high levels of expression not only in muscle but also in hepatocytes. For the liver, this technique required injection directly into the liver vessels (portal vein, hepatic vein, or bile duct) and occlusion of outflow. The present study now demonstrates that high levels of plasmid DNA expression in hepatocytes can be easily obtained by tail vein injections. The highest levels of expression are achieved by rapidly injecting the plasmid DNA in large volumes, ~2.5 ml. This technique has great potential for a wide variety of laboratory studies.
-
high expression of naked plasmid dna in muscles of young rodents
Human Molecular Genetics, 1997Co-Authors: Istvan Danko, Guofeng Zhang, Phillip Williams, Hans Herweijer, Jeffrey S Latendresse, Ildiko Bock, Jon A WolffAbstract:There is a time window at 2 weeks of age for achieving very high levels of Foreign Gene expression from the intramuscular injection of naked plasmid DNA in mice and rats. The highest expression, over 1 microg of luciferase protein/muscle, was obtained in Balb/C mice using constructs containing the CMV promoter, a chimeric intron and the luc+ luciferase Gene. Approximately 50% of the myofibers were intensely blue following the intramuscular injection of a beta-galactosidase expression vector in 2 week old Balb/C mice. The effects of age, mouse strain and construct were multiplicative, resulting in >1000-fold greater luciferase and approximately 20-fold more beta-galactosidase-positive cells. These high levels of expression were unstable and were not observed in larger animals (dog, rhesus monkey). These results indicate that enormous levels of Foreign Gene expression can be obtained in muscle with naked DNA in vivo and will enable the temporary effects of Gene function and expression in rodent muscle to be expeditiously studied.
Frank L Graham - One of the best experts on this subject based on the ideXlab platform.
-
monitoring Foreign Gene expression by a human adenovirus based vector using the firefly luciferase Gene as a reporter
Virus Research, 1993Co-Authors: Suresh K Mittal, Mark R Mcdermott, David C Johnson, Ludvik Prevec, Frank L GrahamAbstract:Abstract We have constructed a helper-independent adenovirus type 5-luciferase recombinant (Ad5-Luc 3) containing the firefly luciferase Gene flanked by simian virus 40 (SV40) regulatory sequences inserted in the early region 3 (E3) of the Ad5 genome. Expression of luciferase in cells infected with Ad5-Luc3 was relatively efficient. In HeLa cells approximately 20 μg luciferase per 106 cells was made by 36 h post-infection and a 62 kilo-Dalton (kDa) luciferase band was clearly visible in a [35S]methionine-labeled Ad5-Luc 3-infected cell extract analyzed directly by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of experiments in which cultured cells were infected with Ad5-Luc 3 in the presence or absence of 1-β- d -arabinofuranosyl cytosine (AraC) showed that the majority of luciferase expression was dependent on viral DNA replication. This suggested that the enzyme was probably translated primarily from mRNA derived from transcripts expressed from the major late promoter of Ad5. An anti-luciferase antibody was raised in a rabbit and used to further characterize the luciferase expressed in HeLa cells infected with Ad5-Luc 3 by immunoprecipitations and Western blot analyses. The half-life of luciferase expressed in HeLa cells infected with Ad5-Luc 3 was calculated to be approximately 6–8 h by pulse chase analysis. Luciferase is likely to be a useful marker for monitoring virus dissemination and Gene expression in experimental animals because assays for enzymatic activity are extremely sensitive and backgrounds are low in all tissues. In mice inoculated intraperitoneally (i.p.) with Ad5-Luc 3, luciferase activity was detected in the liver, spleen, kidney, and lung. A single i.p. inoculation of mice with Ad5-Luc 3 was sufficient to raise anti-luciferase antibody and Ad5 neutralizing antibody which persisted for at least 8 weeks. Even in the presence of circulating anti-luciferase and Ad5 neutralizing antibodies, luciferase activity could be detected in the livers, spleens, and kidneys of mice inoculated i.p. a second time with Ad5-Luc 3.
-
monitoring Foreign Gene expression by a human adenovirus based vector using the firefly luciferase Gene as a reporter
Virus Research, 1993Co-Authors: Suresh K Mittal, Mark R Mcdermott, David C Johnson, Ludvik Prevec, Frank L GrahamAbstract:We have constructed a helper-independent adenovirus type 5-luciferase recombinant (Ad5-Luc 3) containing the firefly luciferase Gene flanked by simian virus 40 (SV40) regulatory sequences inserted in the early region 3 (E3) of the Ad5 genome. Expression of luciferase in cells infected with Ad5-Luc3 was relatively efficient. In HeLa cells approximately 20 micrograms luciferase per 10(6) cells was made by 36 h post-infection and a 62 kilo-Dalton (kDa) luciferase band was clearly visible in a [35S]methionine-labeled Ad5-Luc 3-infected cell extract analyzed directly by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of experiments in which cultured cells were infected with Ad5-Luc 3 in the presence or absence of 1-beta-D-arabinofuranosyl cytosine (AraC) showed that the majority of luciferase expression was dependent on viral DNA replication. This suggested that the enzyme was probably translated primarily from mRNA derived from transcripts expressed from the major late promoter of Ad5. An anti-luciferase antibody was raised in a rabbit and used to further characterize the luciferase expressed in HeLa cells infected with Ad5-Luc 3 by immunoprecipitations and Western blot analyses. The half-life of luciferase expressed in HeLa cells infected with Ad5-Luc 3 was calculated to be approximately 6-8 h by pulse chase analysis. Luciferase is likely to be a useful marker for monitoring virus dissemination and Gene expression in experimental animals because assays for enzymatic activity are extremely sensitive and backgrounds are low in all tissues. In mice inoculated intraperitoneally (i.p.) with Ad5-Luc 3, luciferase activity was detected in the liver, spleen, kidney, and lung. A single i.p. inoculation of mice with Ad5-Luc 3 was sufficient to raise anti-luciferase antibody and Ad5 neutralizing antibody which persisted for at least 8 weeks. Even in the presence of circulating anti-luciferase and Ad5 neutralizing antibodies, luciferase activity could be detected in the livers, spleens, and kidneys of mice inoculated i.p. a second time with Ad5-Luc 3.
Laurence K Grill - One of the best experts on this subject based on the ideXlab platform.
-
heterologous sequences greatly affect Foreign Gene expression in tobacco mosaic virus based vectors
Virology, 1999Co-Authors: Shailaja Shivprasad, Gregory P Pogue, Dennis J Lewandowski, Joann Hidalgo, Jonathan Donson, Laurence K GrillAbstract:Abstract A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient Gene expression were constructed with similar designs but differing in the source of heterologous tobamovirus sequence: Odontoglossum ringspot virus, tobacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame (ORF) and either TMV or heterologous 3′ nontranslated region. The Foreign ORF, from the jellyfish green fluorescent protein (GFP) Gene, was transcribed from the native TMV coat protein subgenomic mRNA promoter, which extended into the coat protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected translational efficiency. However, the major regulatory component of Gene expression in these vectors appeared to be transcriptional rather than translational. There was an inverse relationship between expression of GFP and the heterologous coat protein Genes that was reflected in accumulation of the respective mRNAs and proteins. The most effective vector in this series (30B) contained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3′ nontranslated region from tobacco mild green mosaic virus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble protein in leaves.
Shigetou Namba - One of the best experts on this subject based on the ideXlab platform.
-
Efficient Foreign Gene expression in planta using a plantago asiatica mosaic virus-based vector achieved by the strong RNA-silencing suppressor activity of TGBp1
Archives of Virology, 2014Co-Authors: Nami Minato, Ken Komatsu, Yukari Okano, Kensaku Maejima, Johji Ozeki, Hiroko Senshu, Shuichiro Takahashi, Yasuyuki Yamaji, Shigetou NambaAbstract:Plant virus expression vectors provide a powerful tool for basic research as well as for practical applications. Here, we report the construction of an expression vector based on plantago asiatica mosaic virus (PlAMV), a member of the genus Potexvirus . Modification of a vector to enhance the expression of a Foreign Gene, combined with the use of the foot-and-mouth disease virus 2A peptide, allowed efficient expression of the Foreign Gene in two model plant species, Arabidopsis thaliana and Nicotiana benthamiana . Comparison with the widely used potato virus X (PVX) vector demonstrated that the PlAMV vector retains an inserted Foreign Gene for a longer period than PVX. Moreover, our results showed that the GFP expression construct PlAMV-GFP exhibits stronger RNA silencing suppression activity than PVX-GFP, which is likely to contribute to the stability of the PlAMV vector.