Efficient gene targeting mediated by a lentiviral vector-associated meganuclease
Gene targeting can be achieved with lentiviral vectors delivering donor sequences along with a nuclease that creates a locus-specific double-strand break (DSB). Therapeutic applications of this system would require an appropriate control of the amount of endonuclease delivered to the target cells, a...
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| Format: | Online |
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Oxford University Press
2011
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177226/ |
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pubmed-31772262011-09-21 Efficient gene targeting mediated by a lentiviral vector-associated meganuclease Izmiryan, Araksya Basmaciogullari, Stéphane Henry, Adrien Paques, Frédéric Danos, Olivier Molecular Biology Gene targeting can be achieved with lentiviral vectors delivering donor sequences along with a nuclease that creates a locus-specific double-strand break (DSB). Therapeutic applications of this system would require an appropriate control of the amount of endonuclease delivered to the target cells, and potentially toxic sustained expression must be avoided. Here, we show that the nuclease can be transferred into cells as a protein associated with a lentiviral vector particle. I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein. Integration-deficient lentiviral vectors containing the donor sequences and the I-SceI fusion protein were tested in reporter cells in which targeting events were scored by the repair of a puromycin resistance gene. Molecular analysis of the targeted locus indicated a 2-fold higher frequency of the expected recombination event when the nuclease was delivered as a protein rather than encoded by a separate vector. In both systems, a proportion of clones displayed multiple integrated copies of the donor sequences, either as tandems at the targeted locus or at unrelated loci. These integration patterns were dependent upon the mode of meganuclease delivery, suggesting distinct recombination processes. Oxford University Press 2011-09 2011-06-28 /pmc/articles/PMC3177226/ /pubmed/21715375 http://dx.doi.org/10.1093/nar/gkr524 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Izmiryan, Araksya Basmaciogullari, Stéphane Henry, Adrien Paques, Frédéric Danos, Olivier |
| spellingShingle |
Izmiryan, Araksya Basmaciogullari, Stéphane Henry, Adrien Paques, Frédéric Danos, Olivier Efficient gene targeting mediated by a lentiviral vector-associated meganuclease |
| author_facet |
Izmiryan, Araksya Basmaciogullari, Stéphane Henry, Adrien Paques, Frédéric Danos, Olivier |
| author_sort |
Izmiryan, Araksya |
| title |
Efficient gene targeting mediated by a lentiviral vector-associated meganuclease |
| title_short |
Efficient gene targeting mediated by a lentiviral vector-associated meganuclease |
| title_full |
Efficient gene targeting mediated by a lentiviral vector-associated meganuclease |
| title_fullStr |
Efficient gene targeting mediated by a lentiviral vector-associated meganuclease |
| title_full_unstemmed |
Efficient gene targeting mediated by a lentiviral vector-associated meganuclease |
| title_sort |
efficient gene targeting mediated by a lentiviral vector-associated meganuclease |
| description |
Gene targeting can be achieved with lentiviral vectors delivering donor sequences along with a nuclease that creates a locus-specific double-strand break (DSB). Therapeutic applications of this system would require an appropriate control of the amount of endonuclease delivered to the target cells, and potentially toxic sustained expression must be avoided. Here, we show that the nuclease can be transferred into cells as a protein associated with a lentiviral vector particle. I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein. Integration-deficient lentiviral vectors containing the donor sequences and the I-SceI fusion protein were tested in reporter cells in which targeting events were scored by the repair of a puromycin resistance gene. Molecular analysis of the targeted locus indicated a 2-fold higher frequency of the expected recombination event when the nuclease was delivered as a protein rather than encoded by a separate vector. In both systems, a proportion of clones displayed multiple integrated copies of the donor sequences, either as tandems at the targeted locus or at unrelated loci. These integration patterns were dependent upon the mode of meganuclease delivery, suggesting distinct recombination processes. |
| publisher |
Oxford University Press |
| publishDate |
2011 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177226/ |
| _version_ |
1611476540862758912 |