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Phic31 Integrase as a Promising Tool in Nonviral Gene Therapy

Raphael Liesner

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ISBN 10: 3838123506 / ISBN 13: 9783838123509
Published by Sudwestdeutscher Verlag Fur Hochschulschriften AG
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144 pages. Dimensions: 8.7in. x 5.9in. x 0.3in.Phage-derived C31 integrase represents an attractive tool for site-directed recombination in mammalian cells. Integration is based on recombination between attachment site attB and wtpseudo-attP site. Disadvantages are inefficient nonviral gene delivery and aberrant events (15 chromosomal rearrangementsdeletions) within the host genome. The studys aim was to increase safety and efficiency of C31 integrase. DNA binding domain was mutated by alanine scan, 22 mutants were evaluated for improved integration and intramolecular recombination. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies from 1. 7 to 5. 5-fold. Several mutants showed cell line-dependent integration activities. Excision assays between native attBattP sites revealed 5 mutants with 2-fold enhanced activity. Enhanced recombination between attB and 3 described attP sites (hot spots) in the mammalian genome assumed preferred specificity. 2 mutants showed similar integration activity as wt due to hFIX expression in mouse hepatocytes. Mutational analysis revealed an efficient approach for improvements of integration efficiency in vitro. This item ships from multiple locations. Your book may arrive from Roseburg,OR, La Vergne,TN. Bookseller Inventory # 9783838123509

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Bibliographic Details

Title: Phic31 Integrase as a Promising Tool in ...

Publisher: Sudwestdeutscher Verlag Fur Hochschulschriften AG

Binding: Paperback

Book Condition:New

Book Type: Paperback

About this title

Synopsis:

Phage-derived ?C31 integrase represents an attractive tool for site-directed recombination in mammalian cells. Integration is based on recombination between attachment site attB and wt/pseudo-attP' site. Disadvantages are inefficient nonviral gene delivery and aberrant events (15% chromosomal rearrangements/deletions) within the host genome. The study's aim was to increase safety and efficiency of ?C31 integrase. DNA binding domain was mutated by alanine scan, 22 mutants were evaluated for improved integration and intramolecular recombination. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies from 1.7 to 5.5-fold. Several mutants showed cell line-dependent integration activities. Excision assays between native attB/attP sites revealed 5 mutants with 2-fold enhanced activity. Enhanced recombination between attB and 3 described attP' sites (hot spots) in the mammalian genome assumed preferred specificity. 2 mutants showed similar integration activity as wt due to hFIX expression in mouse hepatocytes. Mutational analysis revealed an efficient approach for improvements of integration efficiency in vitro.

About the Author:

Raphael Liesner studied Biotechnology at the Technical University Berlin and at Technical University of Denmark in Lyngby/ Copenhagen, where he graduated as a M.Sc. He accomplished his Ph.D. in nonviral gene therapy at Max von Pettenkofer-Institute and obtained his doctorate degree in 2010 in Biology at the Ludwig-Maximilians-Universität München.

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