High-Efficient FLPo Deleter Mice in C57BL/6J Background

Abstract

Conditional gene manipulation in mice becomes a routine for genetic studies of mammalian gene functions. Additional site-specific recombinases such as FLP or φ31 provide one more level of gene manipulation flexibility. The recombination activity of the currently available FLP deleter mice remains low. We generated a new FLP deleter mouse line with the mouse codon-optimized FLPo gene in C57BJ/6 background, which showed superior recombination efficacy in comparison to FLPe deleter mice. 100% complete removal of FRT-flanked Neo cassette was observed in all F1 progeny mice carrying both FLPo and Neo cassette, which can be transmitted to F2 generation independent of FLPo activity. Our new FLPo transgenic mice (on pure C57BJ/6 background) will largely facilitate the gene targeting process and is valuable for conditional gene manipulation.

An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase

“…C57Bl/6J fertilized oocytes (Wu et al, 2009). In concordance with our results, one of those PGK-Flpo lines showed complete recombination of FRT-flanked reporters and it was superior to the Flpe deleter line.…”

Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges

“…28 This was independently verified in several contributions demonstrating increased efficiency of Flpo relative to Flpe in deleting sequences flanked by equi-directed FRT. 8,29 Engineering Cre and Flp proteins for nuclear transfer For proteins N45 kDa in size, transport into the nucleus requires specific targeting sequences, called nuclear localization signals (NLS), which mediate interaction with the nuclear transport machinery. 30 It has been suggested that the 38-kDa Cre protein enters the nucleus by passive diffusion, as the addition of a highly efficient NLS from the SV40 large-T antigen did not alter its nuclear localization.…”

Recombinase-mediated cassette exchange (RMCE) and BAC engineering via VCre/VloxP and SCre/SloxP systems

“…A Flp recombinase mutant that is stable at 37C has been developed to improve recombination efficiency at 37C in order to use cultured cells, ES cells, and mice with maximal recombination efficiency (4). VCre originated from a plasmid derived from Vibrio sp., and SCre originated from a plasmid derived from Shewanella sp., ANA-3.…”

“…However, the recombination efficiency of the Flp/FRT system is less than that of the Cre/loxP system. To improve recombination efficiency, some researchers have attempted to optimize Flp by introducing mutations that convert it into a thermostable FLP mutant (3) and by altering its codon usage (4). Additionally, there are currently other genomic engineering tools available such as Dre recombinase (5), which recognizes the 32-bp long rox site, and PhiC31 (6) and R4 (7) integrases.…”