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Efficient Flp-Int HK022 dual RMCE in mammalian cells
Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695500/ https://www.ncbi.nlm.nih.gov/pubmed/23630322 http://dx.doi.org/10.1093/nar/gkt341 |
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author | Voziyanova, Eugenia Malchin, Natalia Anderson, Rachelle P. Yagil, Ezra Kolot, Mikhail Voziyanov, Yuri |
author_facet | Voziyanova, Eugenia Malchin, Natalia Anderson, Rachelle P. Yagil, Ezra Kolot, Mikhail Voziyanov, Yuri |
author_sort | Voziyanova, Eugenia |
collection | PubMed |
description | Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with different target specificities (dual RMCE). Recently, using the Flp–Cre recombinase pair, dual RMCE proved to be efficient, provided the relative ratio of the enzymes during the reaction is optimal. In the present report, we analyzed how the efficiency of dual RMCE mediated by the Flp–Int (HK022) pair depends on the variable input of the recombinases—the amount of the recombinase expression vectors added at transfection—and on the order of the addition of these vectors: sequential or simultaneous. We found that both in the sequential and the simultaneous modes, the efficiency of dual RMCE was critically dependent on the absolute and the relative concentrations of the Flp and Int expression vectors. Under optimal conditions, the efficiency of ‘simultaneous’ dual RMCE reached ∼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE. |
format | Online Article Text |
id | pubmed-3695500 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-36955002013-06-28 Efficient Flp-Int HK022 dual RMCE in mammalian cells Voziyanova, Eugenia Malchin, Natalia Anderson, Rachelle P. Yagil, Ezra Kolot, Mikhail Voziyanov, Yuri Nucleic Acids Res Methods Online Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with different target specificities (dual RMCE). Recently, using the Flp–Cre recombinase pair, dual RMCE proved to be efficient, provided the relative ratio of the enzymes during the reaction is optimal. In the present report, we analyzed how the efficiency of dual RMCE mediated by the Flp–Int (HK022) pair depends on the variable input of the recombinases—the amount of the recombinase expression vectors added at transfection—and on the order of the addition of these vectors: sequential or simultaneous. We found that both in the sequential and the simultaneous modes, the efficiency of dual RMCE was critically dependent on the absolute and the relative concentrations of the Flp and Int expression vectors. Under optimal conditions, the efficiency of ‘simultaneous’ dual RMCE reached ∼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE. Oxford University Press 2013-07 2013-04-27 /pmc/articles/PMC3695500/ /pubmed/23630322 http://dx.doi.org/10.1093/nar/gkt341 Text en © The Author(s) 2013. 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 non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Voziyanova, Eugenia Malchin, Natalia Anderson, Rachelle P. Yagil, Ezra Kolot, Mikhail Voziyanov, Yuri Efficient Flp-Int HK022 dual RMCE in mammalian cells |
title | Efficient Flp-Int HK022 dual RMCE in mammalian cells |
title_full | Efficient Flp-Int HK022 dual RMCE in mammalian cells |
title_fullStr | Efficient Flp-Int HK022 dual RMCE in mammalian cells |
title_full_unstemmed | Efficient Flp-Int HK022 dual RMCE in mammalian cells |
title_short | Efficient Flp-Int HK022 dual RMCE in mammalian cells |
title_sort | efficient flp-int hk022 dual rmce in mammalian cells |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695500/ https://www.ncbi.nlm.nih.gov/pubmed/23630322 http://dx.doi.org/10.1093/nar/gkt341 |
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