Schematic overview of the efficiency of FLPo-mediated RMCE in FlEx gene trap ES cell clones. Shown are all transfected gene trap clone insertions and the corresponding rounded efficiency of exchange reaction of pEx-FLP-dsRed in three different reading frames (0 upper, +1 middle and +2 lowest values in percent, red values indicating the frame matching the gene trap insertion). For the Tardbp insertion, the exchange vector pEx-FLP-hTDP-43(A315T) was used. All gene trap clones used had insertions in an intron of the respective gene, and the ratio on the right indicates in which intron out of how many total introns the insertion is located. On the left, the size of the genes is indicated in kilobases, and the total size of the genes is not drawn in scale, with omitted DNA indicated. Untranslated and translated exons according to Ensembl. MGI Gene Identifiers and absolute expression levels in ES cells are listed in Supplementary Table S1.

In vivo excision of the hygromycin-resistance cassette. (A) The gene trap vector rFLPRosabetageo, which was inserted in intron 1 of the mouse Tardbp gene (clone D045A10 in Fig. 2), was replaced by pEx-FLP-hTDP-43(A315T). A mutant mouse line with this genomic modification was established and crossed with Rosa26-Cre deleter mice. (B) The RMCE allele in the mouse Tardbp intron 1 after Cre-mediated excision of the hygromycin-resistance cassette. (C) Left: total protein was extracted from embryonic heads (lanes 1–3), after mating to Cre-deleter mice at E17.5 or HEK293 cells (positive control, lane 4). Three western blots were done in parallel using antibodies against human Tdp-43 (hTDP-43), human and mouse TDP-43 (mTDP-43) and β-actin. TDP-43 runs at ∼45 kDa, β-actin runs at 42 kDa. Right: DNA for genotyping was obtained from tails. PCR was performed using the primers SR/TP or B048/B045 as depicted in (A). The presence of Cre was detected using primers specific for Cre-recombinase. Exc: excision (of the hygromycin-resistance cassette); Ret: retention (of the hygromycin-resistance cassette). LTR: long terminal repeat, SA: splicing acceptor, PGK-pA: phosphoglycerate kinase poly A signal; BGHpA: bovine growth hormone polyA signal; attB1/2: gateway clonase recognition sites.

(A) Schematic outline of the procedure for individual modification and use of pEX-FLP. Any DNA sequence, which is cloned between the attL1/2 recombination sites in the pENTR-EX, can be exchanged with an attR1/2 flanked cat ccdb cassette of pEX-Dest in vitro. (B) Schematic illustration of the FLPo RMCE in ES cells. pEX-FLP harbors two heterospecific flippase recombination sites, therefore cotransfection of FLPo and pEX-FLP to any FlEx gene trap ES cell clone allows replacement of β-geo cassette. (C) Analysis of successfully exchanged locus with pEX-FLP-dsRed containing a adSA-dsRed-BGHpA sequence flanked by IR/DR of Sleeping Beauty transposase. A representative PCR screen of seven hygromycin resistant clones (E307D01 derivates, A01–A07) concerning orientation of the genetrap vector and successful exchange (left: primers B045, B048 and B050; right: primers SR, TP). A01 and A06 are inverted β-geo insertions and show an 800-bp band on the left and no band on the right. A02–A05 are successfully exchanged clones and show an 839-bp band on the left and the small 239-bp band on the right. Clone A07 carries the original β-geo insertion and gives rise to the 631-bp band on the left and the 516-bp band on the right.

(A) Removal of hygromycin resistance upon Cre activity. Cre-transfected exchange clone A03 (E307D01/pExFLP-dsRed derivative) was analysed using primers located within both the hygromycin/dsRed cassettes (TP) and the LTR of the original gene trap vector (SR). PCR on total genomic DNA of five pooled transfected dishes (lanes 1–5) compared to genomic DNA of clone A03 before Cre transfection (A03 −Cre). The internal primer (TP) gives rise to the larger product (619 bp) after successful excision of the hygromycin cassette. Before hygromycin excision, primer TP amplifies a smaller product of 239 bp. (B) RT-PCR and triple PCR analysis to determine splicing to the dsRed after hygromycin excision by using external primers in exon II (5) of the insertion locus (Msi2) and an internal dsRed primer (I) and as internal wild-type control a primer located in exon III (3) of the Msi2 gene. cDNA of individual clones derived from Cre-transfected clone A03 after puromycin selection (lanes labelled 1–3) compared to cDNA of clone A03 before Cre transfection (lane A03 −Cre). As control genomic DNA of two gene trap clones (E307D01 and E326E12) and pEX-FLP-dsRed plasmid was used. (C) Western blot analysis of protein extracted from ES cells probed with a polyclonal RFP antibody. As controls wild-type ES cells either untransfected (WT) or transiently transfected with two different dsRed expression plasmids (WT + RFPI + II) were used. Exchange clones A03 and D04 were analysed prior (−CRE) and after (+CRE) transient CRE transfection. DsRed protein positive samples show a 27-kDa band. Lower blot shows loading control with beta actin monoclonal antibody (42 kDa).

PCR analysis of SB100 transfected exchange clone to determine mobilization of IR/DR flanked cassette. Genomic DNA of pooled SB100 transfected dishes (0, 10 and 70-µg SB100 plasmid) of exchange clone A03 (E307D01/pExFLP-dsRed derivative) was screened with primers located 5′ of the F3 site (B045), in the pA of dsRed (B048) and in the hygromycin-resistance cassette (H). DNA was screened with either primer combination H/B045 or B048/B045, as controls genomic DNA of clone A03 (A03 −SB) and E307D01 was used. Primers H/B045 yielded a 1-kb product after successful excision of the IR/DR flanked cassette. Without mobilization, a 839-bp band was amplified by primers B048/B045, whereas the original gene trap insertion led to a 631-bp band.