Abstract
(English)
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1. Generation of a transgenic mouse that expresses a neoself antigen (LCMV GP) in a tissue-specific and inducible way.
This project is dedicated to the generation of a transgenic mouse line that expresses LCMV-GP in a tissue-specific (oligodendrocytes, pancreatic beta cells, B cells, dendritic cells) and inducible way. This mouse will be useful in answering the following questions: 1) Are different peripheral tissues differently susceptible to autoimmune attack?, 2) Are different kinds of immune responses (acute, memory, helper CTL) differently induced/tolerised by peripheral organs (oligodendrocytes, beta cells) or antigen presenting cells (APC, B cells, dendritic cells)?, 3) Do different infectious agents expressing LCMV GP induce qualitatively and quantitatively different immunity resulting in different capacity to induce autoimmunity?, and 4) What is the contribution of epitope spreading to autoimmunity?
We generated the following tools. A) Recombinant Listeria monocytogenes expressing LCMV GP, B) Different recombinant Vaccinia Viruses expressing LCMV helper/and or CTL epitopes, C) A transgenic mouse line expressing LCMV-GP + eGFP (as a marker protein) under control of the ubiquitous human ubiquitinC promoter. Constitutive expression of the transgene is inhibited by a floxed STOP cassette between promoter and transgene. After ubiquitous, Cre-mediated removal of the STOP cassette, these mice were shown to be immunologically tolerant for LCMV-GP, demonstrating expression of the transgene only after removal of the STOP cassette. We now have two independent founder lines with different expression level, D) Mouse lines expressing inducible Cre (by making it a fusion protein with the ligand binding domain of the mutated human estrogen receptor ER) in oligodendrocytes, in beta cells of the pancreas and in B cells. Expression is induced in vivo by administration of tamoxifen. The construct that drives dendritic cell-specific expression is being injected at the moment.
Crossing the STOP-LCMV-GPeGFP mouse with the CreER mouse, followed by injection of tamoxifen induces at a chosen timepoint expression of LCMV-GP in a tissue of choice.
2. Generation of conditional tissue ablation mice based on the Cre/LoxP system
We will generate a binary transgenic system that enables us to selectively eliminate specific cell types by the inducible expression of the highly toxic diphtheria toxin a (DTA). We will start with eliminating macrophage subpopulations to investigate their role in the induction of immunity, immunopathology and autoimmunity. Our system is designed such that any other cll type can be ablated after minor changes in the transgene constructs.
We generated the following tools. A) A mouse expressing DTA under control of the ubiquitous ubiquitin C promoter, but only after removal of the floxed STOP cassette (see above) which cloned between promoter and DTA. We obtained 4 founders that transmit the transgene to their offspring, and that are now being further analysed. B) A mouse expressing DTA under control of the mouse actin promoter, but only after removal of floxed eGFP, which is cloned between promoter and DTA. The expression of eGFP will facilitate the selection of transgenic offspring. Of this line, we obtained 15 founders, of which 4 were selected for further analysis. These mice are now being bred with LysM-Cre transgenic mice to give rise to macrophage-deficient offspring.
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