Supplementary Figure 11: Reversible conditional gene inactivation with FLIP-FlpE (FLIP-Flp Excision) intronic cassette. | Nature Methods

Supplementary Figure 11: Reversible conditional gene inactivation with FLIP-FlpE (FLIP-Flp Excision) intronic cassette.

From: One-step generation of conditional and reversible gene knockouts

Supplementary Figure 11

(a) The FLIP-FlpE cassette containing a DsRed reporter gene was inserted into the cDNA of eGFP as an artificial intron and transfected in HEK 293 cells. The FLIP-FlpE cassette contains the same elements as the FLIP cassette except the addition of two FRT sites flanking the region containing the cryptic splice acceptor and pA. SD, splice donor; SA1, SA2, splice acceptor; purple triangles, LoxP1; pink triangles, Lox5171; FRT sites, yellow ovals; BP1, BP2 (blue circles), branching point; pA, polyadenylation signal.

(b) Following insertion, the cassette functions as an intron and does not disrupt the expression of the eGFP cDNA. Hence, both eGFP and DsRed proteins are expressed (top row). After Cre recombination the eGFP expression is disrupted, and only DsRed expression is maintained (bottom row). Following Flp recombination, the mutagenic cassette is excised and the eGFP expression is restored.

(c) The FLIP-FlpE cassette containing a resistance gene is inserted into the exon 5 of Ctnnb1. SD, splice donor; SA, splice acceptor; pink and purple triangles, loxP site; FRT sites, yellow ovals; Blue circles, branching point.

(d) PCR detection of FLIP-FlpE cassette insertion in the Ctnnb1 locus of mouse embryonic stem cells. The correctly targeted clone (H11) is positive for 5’ and 3’arm genotyping PCR reactions and it is homozygous.

(e) Representative bright field images of the H11 clone in control, Cre induction and Cre and Flp dual induction. Scale bar 400 μm.

(f,g) Detection of β-catenin protein by immunofluorescence (f) and western blotting (g) in control, Cre induction and Cre and Flp dual induction. Scale bar 100 μm for left and right panels, scale bar 200 μm for the middle panel. Full blot(s) can be seen in Supplementary Fig. 14f.

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