Efficient Flp-Int HK022 dual RMCE in mammalian cells.
Nucleic Acids Res
; 41(12): e125, 2013 Jul.
Article
en En
| MEDLINE
| ID: mdl-23630322
ABSTRACT
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 â¼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Recombinación Genética
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Integrasas
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ADN Nucleotidiltransferasas
Límite:
Animals
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2013
Tipo del documento:
Article
País de afiliación:
Estados Unidos