TY - JOUR
T1 - Efficient Flp-Int HK022 dual RMCE in mammalian cells
AU - Voziyanova, Eugenia
AU - Malchin, Natalia
AU - Anderson, Rachelle P.
AU - Yagil, Ezra
AU - Kolot, Mikhail
AU - Voziyanov, Yuri
N1 - Funding Information:
National Institutes of Health (NIH) [R01GM085848 to Y.V.]; German Israeli Foundation for Scientific Research and Development (G.I.F) [1062-62.3/2008]; Israel Science Foundation [702/11 to E.Y. and M.K.]. Funding for open access charge: NIH.
PY - 2013/7
Y1 - 2013/7
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84880212923&partnerID=8YFLogxK
U2 - 10.1093/nar/gkt341
DO - 10.1093/nar/gkt341
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AN - SCOPUS:84880212923
SN - 0305-1048
VL - 41
SP - e125
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 12
ER -