N-ethyl-N-nitrosourea-based generation of mouse models for mutant G protein-coupled receptors.

Chemical random mutagenesis techniques with the germ line supermutagen N-ethyl-N-nitrosourea (ENU) have been established to provide comprehensive collections of mouse models, which were then mined and analyzed in phenotype-driven studies. Here, we applied ENU mutagenesis in a high-throughput fashion for a gene-driven identification of new mutations. Selected members of the large superfamily of G protein-coupled receptors (GPCR), melanocortin type 3 (Mc3r) and type 4 (Mc4r) receptors, and the orphan chemoattractant receptor GPR33, were used as model targets to prove the feasibility of this approach.

Efficient and fast targeted production of murine models based on ENU mutagenesis.

Mice with targeted genetic alterations are the most effective tools for deciphering organismal gene function. We generated an ENU-based parallel C3HeB/FeJ sperm and DNA archive characterized by a high probability to identify allelic variants of target genes as well as high efficiencies in allele retrieval and model revitalization. Our archive size of over 17,000 samples contains approximately 340,000 independent alleles (20 functional mutations per individual sample).

Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry.

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation.

Reliable recovery of inbred mouse lines using cryopreserved spermatozoa.

Since the mouse has become the most detailed model system to investigate the genetics and pathogenesis of human diseases, large numbers of new mouse strains have and continue to be produced. In nearly all animal facilities, the maintenance of breeding colonies is limited and mouse strains have to be archived in an efficient way. This study was undertaken to test the reliability of recovering mouse lines by use of cryopreserved spermatozoa from individual male mice.

The resistance against Listeria monocytogenes and the formation of germinal centers depend on a functional death domain of the 55 kDa tumor necrosis factor receptor.

The biological functions mediated by the death domain of the 55-kDa tumor necrosis factor receptor (TNFRp55) in vivo are still elusive. TNFRp55 mutants lacking a functional death domain were expressed in TNFRp55-/- and in TNFRp55+/- mice as transgenes under control of the H-2Kb promoter. Analysis of these animals revealed that signals originating from the TNFRp55 death domain are indispensable for the protection against Listeria monocytogenes, the expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the spleen and the development of splenic germinal centers.