دورية أكاديمية
Kinetics and longevity of ΦC31 integrase in mouse liver and cultured cells.
| العنوان: | Kinetics and longevity of ΦC31 integrase in mouse liver and cultured cells. |
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| المؤلفون: | Chavez CL; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA., Keravala A, Woodard LE, Hillman RT, Stowe TR, Chu JN, Calos MP |
| المصدر: | Human gene therapy [Hum Gene Ther] 2010 Oct; Vol. 21 (10), pp. 1287-97. |
| نوع المنشور: | Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: M.A. Liebert Country of Publication: United States NLM ID: 9008950 Publication Model: Print Cited Medium: Internet ISSN: 1557-7422 (Electronic) Linking ISSN: 10430342 NLM ISO Abbreviation: Hum Gene Ther Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: New York : M.A. Liebert, c1990- |
| مواضيع طبية MeSH: | Plasmids* , Transfection*, Integrases/*genetics , Integrases/*metabolism , Liver/*enzymology, Animals ; Attachment Sites, Microbiological/genetics ; Blotting, Southern ; Blotting, Western ; Cell Line ; Fluorescent Antibody Technique ; Gene Expression ; Gene Silencing ; Genetic Therapy ; Genetic Vectors ; HeLa Cells ; Humans ; Kinetics ; Mice ; Mice, Inbred C57BL ; Polymerase Chain Reaction ; Recombination, Genetic ; Time Factors |
| مستخلص: | 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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| معلومات مُعتمدة: | T32 CA009302 United States CA NCI NIH HHS; HL68012 United States HL NHLBI NIH HHS; CA09302 United States CA NCI NIH HHS |
| المشرفين على المادة: | EC 2.7.7.- (Integrases) |
| تواريخ الأحداث: | Date Created: 20100526 Date Completed: 20110321 Latest Revision: 20211020 |
| رمز التحديث: | 20231215 |
| مُعرف محوري في PubMed: | PMC2974851 |
| DOI: | 10.1089/hum.2010.049 |
| PMID: | 20497035 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1557-7422 |
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| DOI: | 10.1089/hum.2010.049 |