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. 2010 Jan;7(1):50-2.
doi: 10.1038/nmeth.1406. Epub 2009 Dec 6.

Floxin, a resource for genetically engineering mouse ESCs

Veena Singla  1 , Julie Hunkapiller, Nicole Santos, Allen D Seol, Andrew R Norman, Paul Wakenight, William C Skarnes, Jeremy F Reiter
Affiliations

Floxin, a resource for genetically engineering mouse ESCs

Veena Singla et al. Nat Methods. 2010 Jan.

Abstract

We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations were reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable high-throughput modification of many genes in mouse ESCs.

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Figures

Figure 1
Figure 1. The Floxin strategy for reversion and modification of gene trap loci
(a) In the wild type allele of Your Favorite Gene (YFG), exons 1 and 2 are spliced together and translated to produce full length YFG protein. In the gene trap line of genotype YFGGt/+, a splice acceptor (yellow box) flanked by Lox71 and LoxP sites co-opts splicing to create a fusion between βgeo and the 5’ endogenous exon. βgeo encodes a fusion of β-galactosidase and neomycin resistance. (b) Reversion of the gene trap mutation to yield revertant line YFGRev (genotype YFPRev/+). Cre recombines the Lox71 and LoxP sites, restoring endogenous splicing and wild type expression of YFG. (c) Vectors for Floxin-mediated cassette insertion. Cre recombines the genomic Lox71 and Floxin Lox66 sites to integrate the pFloxin construct into the genomic locus. In these examples, Floxin mediates insertion of exon 2 of YFG with a C-terminal Flag tag (left) or the full-length cDNA for YFG with an N-terminal HA tag (right). (d) Schematic showing Floxin alleles that express tagged YFG under the control of endogenous regulatory elements and re-express βgeo. (e) Immunoblots with the indicated antibodies ESCs of the Ofd1Gt genotype. 10 µg protein per lane.
Figure 2
Figure 2. Efficient reversion of gene trap mutations and Floxin-mediated engineering of new alleles
(a) Plot of β-galactosidase activity in cell lines of the indicated genotypes. Error bars are standard deviations from 1–4 different experiments with a minimum of 3 replicates each. (b) Immunoblot showing Suz12 (left and middle panels, 20 µg protein per lane) and Sall4 (right panel, immunoprecipitated from 500 µg total protein for each cell line) protein levels in the cell lines of the indicated genotypes. (c) Immunoblots showing expression of full length Myc-tagged Ofd1 (left panel, 25 µg protein per lane; right panel, immunoprecipitate from 800 µg total protein for each cell line), and of Ofd1 in Ofd1Rev cells (middle panel, immunoprecipitate from 4 mg total protein for each cell line). (d, e) Representative fluorescence micrographs of cell lines of the indicated genotypes. Cilia (acetylated Tubulin, green), centrosomes (γ-Tubulin, red), DNA (DAPI, blue). (f) Representative fluorescence micrograph of Ofd1-Myc localization. Ofd1 (Myc, green), centrosome (Centrin, red), DNA (DAPI, blue). (g) Representative fluorescence micrograph of Suz12-TAP localization. Suz12 (Flag, green), centrosome (Acetylated Tubulin, red), DNA (DAPI, blue). (h) Immunoblot showing wild type Suz12 and Suz12-TAP protein downregulation upon differentiation of Suz12Suz12TAP/+ cells. 3 µg protein per lane. Scale bars 5 µm, with magnified inset scale bar 1 µm.
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