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Honolulu, HI, United States

Urschitz J.,Biochemistry and Physiology | Kawasumi M.,Biochemistry and Physiology | Kawasumi M.,Keio University | Owens J.,Biochemistry and Physiology | And 13 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flankedby terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches. Source

Urschitz J.,Biochemistry and Physiology | Owens J.B.,Biochemistry and Physiology | Moisyadi S.,Biochemistry and Physiology
Ultrasound in Medicine and Biology | Year: 2013

Our aim was to evaluate the delivery of transposase-based vectors by ultrasound targeted microbubble destruction (UTMD) in mice. DNA vectors were attached to cationic lipid microbubbles (1-3μm in diameter), injected intravenously and delivered to the liver by destruction of the carrier bubbles with ultrasound in burst mode at 1.0MHz, 20-μs pulse duration, 10-Hz pulse repetition frequency and ~1.3-MPa acoustic peak negative pressure. We evaluated the expression and genomic integration of conventional (pcDNA3) and piggyBac transposase-based (p. mGENIE) reporter vectors. Invivo, we observed UTMD-mediated liver-specific expression of p. mGENIE for an average of 24d, compared with 4d with pcDNA3. Reporter expression was located predominately near blood vessels initially, whereas expression after 3d was more evenly distributed through the parenchyma of the liver. We confirmed random genomic integration for p. mGENIE invitro; however, integration events for p. mGENIE invivo were targeted to specific areas of chromosome 14. Our results suggest that a combination of UTMD and non-viral DNA transposase vectors can mediate weeks of hepatic-specific gene transfer invivo, and analyses performed by non-restrictive linear amplification-mediated (nrLAM) polymerase chain reaction, cloning and sequencing identify an unexpected tropism for integration within a specific sequence on chromosome 14 inmice. UTMD delivery of transgenes may be useful for the treatment of hepatic gene deficiency disorders. © 2013 World Federation for Ultrasound in Medicine & Biology. Source

Prusty A.K.,CIFE | Kohli M.P.S.,CIFE | Sahu N.P.,Biochemistry and Physiology | Pal A.K.,Biochemistry and Physiology | And 3 more authors.
Pesticide Biochemistry and Physiology | Year: 2011

Experiment was carried out to determine the median lethal concentration (LC50) of fenvalerate to Labeo rohita fingerlings. After determining the LC50 value of fenvalerate, a sub-lethal concentration (1/3rd of LC50) of fenvalerate was exposed for 15days. Significant alterations in SOD (P<0.05) activity of liver and gill was observed due to fenvalerate. Catalase activity in gills of fishes was also affected significantly (P<0.05). WBC, NBT and Hct values were reduced significantly in fenvalerate exposed fishes as compared to control group, whereas blood glucose level showed higher values in fenvalerate exposed group. Serum total protein and albumin were also reduced significantly as a result of fenvalerate exposure. Significant increase in the serum GOT, serum GPT, creatinine, triglyceride and serum ACP was noticed after 15days of fenvalerate exposure. Results indicated that short term exposure of fenvalerate can induce biochemical and haematological alterations causing stress to L. rohita fingerlings. © 2011 Elsevier Inc. Source

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