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A Flexible Toolkit for Controlling Gene Expression in the Mouse

FLPFLEX. A Flexible Toolkit for Controlling Gene Expression in the Mouse SPECIFIC TARGETED RESEARCH OR INNOVATION PROJECT Thematic Priority 1.1.1 Life sciences, genomics and biotechnology for health Proposal/Contract no.: 513769 Grant duration: 7/2005 - 6/2008. FLPFLEX: Partners.

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A Flexible Toolkit for Controlling Gene Expression in the Mouse

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  1. FLPFLEX A Flexible Toolkit for Controlling Gene Expression in the Mouse SPECIFIC TARGETED RESEARCH OR INNOVATION PROJECT Thematic Priority 1.1.1Life sciences, genomics and biotechnology for health Proposal/Contract no.: 513769 Grant duration: 7/2005 - 6/2008

  2. FLPFLEX: Partners Nadia Rosenthal EMBL Andrea Ballabio TIGEM, Italy Wolfgang Wurst GSF, Germany Riccardo Cortese IRBM, Italy Hansjörg Hauser GBF, Germany

  3. FLPFLEX: Rationale • Mammalian system complexity: • Many cell types function in each tissue • Many tissue types function in each organ • Many organs function in each system • Many stage-specific functions in each disease • Our control of mouse gene expression in space and time needs better resolution for modelling human disease

  4. FLPFLEX: Fulfilling the need for higher resolution To meet the increasing need for tissue-specific and/or regulated gene expression for multiple applications in the mouse.

  5. FLPFLEX: Mission • FLPFLEX is dedicated to the development of novel tools to manipulate gene expression: • for dissecting complex genetic pathways • for generating more accurate models of • human disease.

  6. FLPFLEX: Approach • FLPFLEX cassette exchange allows multiple rapid manipulations in a single genomic locus: • by engaging the cell’s own transcriptional regulatory circuits to capture novel expression patterns • by providing powerful and adaptable tools for designing increasingly complex genetic models of human physiology and pathology

  7. Conventional gene trapping SA SA SA LacZ LacZ LacZ Neo Neo Neo pA pA pA Splicing ATG mRNA ex1 Lacz Fusion Protein (Reporter) Neo ATG x P ex1 ex2 ex3 pA Endogenous locus geo Vector Trapped allele ATG P ex1 ex2 ex3 pA No further modification possible

  8. Limitation of conventional gene trapping P ex1 ex2 ex3 pA Interesting trapped locus P SA LacZ Neo pA ex1 ex2 ex3 pA P transactivator ex3 pA P toxin ex3 pA P Cre ex3 pA P Gene of interest ex3 pA For each construct, an additional homologous recombination must be performed in the trapped locus

  9. FLPFLEX: flexible gene switching F F5 Heterotypic FLP target sequences F5 F Gene of interest + FlpE P F Gene of interest ex1 F5 ex2 ex3 pA FLPFLEX allele ATG P SA Reporter Selector pA ex1 ex2 ex3 pA Gene of interest: - Cre - transactivator - toxin - knock-in F One trapped locus, multiple gene readouts with rapid enzymatic recombination

  10. FLPFLEX tools Tissue-specific reversible control of gene expression Tissue-specific TET Tissue-specific conditional knock-out/knock-in Tissue-specific Cre Tissue-specific cell ablation Tissue-specific Toxin Tissue-specific knock-down Tissue-specific RNAi Tissue-specific oncogenesis Tissue-specific Ras, etc.. Aim Tool

  11. A. Generation of FLPFLEX ES cell clones F F5 SA ATG SA LacZ Neo pA FlipRosaβgeo (EUCOMM) SA ES cells + vector Electroporation Selection in G418 & picking Growing colonies to confluency Storage of master plate bgalactivity

  12. B. Selection of FLPFLEX ES cell clones 5000 integration sites gal positive FLPFLEX clones FISH analysis mRNA Extraction 5’ RACE DNA sequencing, in silico analysis Automated ISH of adult mouse tissues Selection of expression Pattern of interest

  13. C. Generation of FLPFLEX mice 20 characterised mouse lines Injection of gal-positive FLPFLEX ES cell into mouse blastocyst Chimera production Germline transmission Analysis of gal expression patterns

  14. FLPFLEX proof of concept: GFP cassette exchange Trapped gene expression GFP reporter expression In situ hybridisation GFP is flipped into in FLFPEX targeted locus in ES clone GFP reporter accurately reflects trapped gene expression pattern Trapped gene regulation is not perturbed by cassette exchange

  15. FLPFLEX: Key features Multifunctional readout vectors A library of expression patterns C) Automated high-throughput screening for clones D) Automated high throughput expression analysis E) Flexible disease modeling in mice F) FLPFLEX public database (with EUCOMM) G) Dissemination of material

  16. FLPFLEX: A complement to EUCOMM Harmonised vectors Complementary set of targeted genes C) Emphasis on useful expression patterns D) Rapid proof of concept E) Pipeline to Cre Zoo

  17. FLPFLEX

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