Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination.

AUTOR(ES)

Buchholz, F

RESUMO

Genomic manipulations using site-specific recombinases rely on their applied characteristics in living systems. To understand their applied properties so that they can be optimally deployed, we compared the recombinases FLP and Cre in two assays. In both Escherichia coli and in vitro, FLP shows a different temperature optimum than Cre. FLP is more thermolabile, having an optimum near 30 degrees C and little detectable activity above 39 degrees C. Cre is optimally efficient at 37 degrees C and above. Consistent with FLP thermolability, recombination in a mammalian cell line mediated by a ligand- regulated FLP-androgen receptor fusion protein is more efficient at 35 degrees C than at higher temperatures. We also document a mutation in a commercially available FLP plasmid (FLP-F70L) which renders this recombinase even more thermolabile. The different temperature optima of FLP, FLP-F70L and Cre influence their strategies of usage. Our results recommend the use of Cre for applications in mice that require efficient recombination. The thermolabilities of FLP and FLP-F70L can be usefully exploited for gain of function and cell culture applications.

Documentos Relacionados

• Site-specific recombinases: changing partners and doing the twist.
• Characterization of Holliday structures in FLP protein-promoted site-specific recombination.
• Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination.
• Site-specific cleavage of chromosomes in vitro through Cre-lox recombination.
• Holliday intermediates and reaction by-products in FLP protein-promoted site-specific recombination.