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Kinetics and longevity of ΦC31 integrase in mouse liver and cultured cells.
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Kinetics and longevity of ΦC31 integrase in mouse liver and cultured cells.

Author: CL Chavez Affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.A KeravalaLE WoodardRT HillmanTR StoweAll authors
Edition/Format: Article Article : English
Publication:Human gene therapy, 2010 Oct; 21(10): 1287-97
Other Databases: WorldCatWorldCatWorldCat
Summary:
The ΦC31 integrase system provides genomic integration of plasmid DNA that may be useful in gene therapy. For example, the ΦC31 system has been used in combination with hydrodynamic injection to achieve long-term expression of factor IX in mouse liver. However, a concern is that prolonged expression of ΦC31 integrase within cells could potentially stimulate chromosome rearrangements or an immune response. Western  Read more...
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Document Type: Article
All Authors / Contributors: CL Chavez Affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.; A Keravala; LE Woodard; RT Hillman; TR Stowe; JN Chu; MP Calos
ISSN:1043-0342
Language Note: English
Unique Identifier: 672347824
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Abstract:

The ΦC31 integrase system provides genomic integration of plasmid DNA that may be useful in gene therapy. For example, the ΦC31 system has been used in combination with hydrodynamic injection to achieve long-term expression of factor IX in mouse liver. However, a concern is that prolonged expression of ΦC31 integrase within cells could potentially stimulate chromosome rearrangements or an immune response. Western blot and immunofluorescence analyses were performed to investigate the duration of ΦC31 integrase expression in mouse liver. Integrase was expressed within 2 to 3 hr after hydrodynamic injection of a plasmid expressing ΦC31 integrase. Expression peaked between 8 and 16 hr and fell to background levels by 24-48 hr postinjection. Analysis of the amount of integrase plasmid DNA present in the liver over time suggested that the brief period of integrase expression could largely be accounted for by rapid loss of the bulk of the plasmid DNA, as well as by silencing of plasmid expression. PCR analysis of integration indicated that ΦC31 integrase carried out genomic integration of a codelivered attB-containing plasmid by 3 hr after plasmid injection. Integrase was expressed for longer times and at higher levels in transfected cultured cells compared with liver. Inhibitor studies suggested that the enzyme had a short half-life and was degraded by the 26S proteasome. The short duration of integrase expression in liver and rapid integration reaction appear to be features favorable for use in gene therapy.

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