German Genetrap Consortium (GGTC)
Large scale systematic mutagenesis in mouse embryonic stem (ES) cells is most effectively induced using gene trap vectors, a class of promoter-less reporter/selector gene cassettes integrating randomly into a large collection of chromosomal sites. We have established the largest conditional gene trap library worldwide which is available to the academic community. During this project, we have also generated mutations in 791 loci which are associated to human disease. Gene trap mutations are highly mutagenic and approximately 95% of the integrations lead to loss-of-function mutations. Our mutant ES cell resource is available on the German Gene Trap Consortium.
More recently, we have implemented further developments of multipurpose alleles, that allow post-trapping modifications by means of recombinase-mediated cassette exchange (RMCE). This allows us for example to replace mouse genes with their human wildtype or mutant counterparts in order to express these genes in the right cell at the right time and therefore to try to model human disease. We are currently actively applying this technique in order to generate mouse models for a number of neurodegenerative diseases ("Animal models for neurodegenerative disease (Morbus Alzheimer, Frontotemporal Dementia and Amytrophic Lateral Sclerosis")
- Figure 1: (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 co-transfection 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 8 hygromycin resistant clones (E307D01 derivates, A01-A07) concerning orientation of the genetrap vector and successful exchange (left: primers B045, B048, 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.
- 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 b-actin. TDP-43 runs at approximately 45 KDa, b-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.
Most important publications:
Frank Schnütgen, Franziska Ehrmann, Ina Poser, Nina C. Hubner, Jens Hansen, Thomas Floss, Wolfgang Wurst, Anthony Hyman, Matthias Mann, and Harald von Melchner (2011) Use of public gene trap resources for high throughput proteome analysis. Nature Methods, 8(2):103-104.
Schebelle L, Wolf C, Stribl C, Javaheri T, Schnütgen F, Ettinger A, Ivics Z, Hansen J, Ruiz P, von Melchner H, Wurst W, Floss T. (2010) Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps. Nucleic Acids Res. 38(9):e106.
Floss T, Schnütgen (2008) Conditional gene trapping using the FLEx system. F.Methods Mol Biol. 435:127-38.
Hansen J, Floss T, van Sloun P, Füchtbauer EM, Arnold HH, Vauti F, Schnütgen F, Wurst W, von Melchner H, Ruiz P (2003) A large scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome. Proc Nat Acad Sci (USA) 100, 9918-9922.
Group members:
| Thomas Floss Coordination of requests | +49 89 3187 2887 | floss@helmholtz-muenchen.de |
| Jens Hansen Bioinformatics | +49 89 3187 1193 | hansen@helmholtz-muenchen.de |
| Denise Herold Quality Controls and clone distribution | +49 89 3187 3825 | denise.herold@helmholtz-muenchen.de |