Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters.

Research on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) in the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project as parameters for the generation of novel animal models for human diseases. The analysis was carried out on more than 16,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the levels of the chosen parameters. Identification of animals exhibiting altered values and transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters MCV, RBC, and PLT. Analysis of the causative mutation was started in selected lines, thereby revealing a novel mutation in the transferrin receptor gene (Tfrc) in one line. Thus, novel phenotype-driven mouse models were established to analyze the genetic components of hematological disorders.

An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice.

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.

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. Parallel archives of DNA and sperm from mice mutagenized with ENU were screened for mutations in these GPCR, and in vitro assays served as a preselection step before in vitro fertilization was performed to generate the appropriate mouse model. For example, mouse models for inherited obesity were established by selecting fully or partially inactivating mutations in Mc4r. Our technology described herein has the potential to provide mouse models for a GPCR dysfunction of choice within <4 mo and can be extended to other gene classes of interest.

Use of an exon-trapping vector for the evaluation of splice-site mutations.

Prediction of the effects of splice-site variations by sequence analysis is difficult. In this study we provide the means for a rapid evaluation of the potential for splice-site mutations to interfere with RNA processing. The system may be useful in reverse genetics or mapping studies when isolation and characterization of mRNA is arduous or not possible. In the assay we cloned wild-type and mutant sequences of murine splice-site mutations into an exon-trapping vector and characterized splicing of both recombinant transcripts in a transient cell culture system. Results from this artificial assay were compared with in vivo data from the respective mouse models. We found that the exon-trapping system allows one to confidently predict whether a splice-site variation is going to have a splicing effect in vivo, but the system does not always reflect in vivo splicing in detail. In summary, the exon-trapping system is a reliable and easy-to-use tool for a first evaluation of splice effects.

Identification and regulation of cold-inducible factors of Bordetella bronchiseptica.

The expression of bacterial cold-shock proteins (CSPs) is highly induced in response to cold shock, and some CSPs are essential for cells to resume growth at low temperature. Bordetella bronchiseptica encodes five CSPs (named CspA to CspE) with significant amino acid homology to CspA of Escherichia coli. In contrast to E. coli, the insertional knock-out of a single csp gene (cspB) strongly affected growth of B. bronchiseptica independent of temperature. In the case of three of the csp genes (cspA, cspB, cspC) more than one specific transcript could be detected. The net amount of cspA, cspB and cspC transcripts increased strongly after cold shock, while no such effect could be observed for cspD and cspE. The exposure to other stress conditions, including translation inhibitors, heat shock, osmotic stress and nutrient deprivation in the stationary phase, indicated that the csp genes are also responsive to these conditions. The coding regions of all of the cold-shock genes are preceded by a long non-translated upstream region (5'-UTR). In the case of the cspB gene, a deletion of parts of this region led to a significant reduction of translation of the resulting truncated transcript, indicating a role of the 5'-UTR in translational control. The cold-shock stimulon was investigated by 2D-PAGE and mass spectrometric characterization, leading to the identification of additional cold-inducible proteins (CIPs). Interestingly, two cold-shock genes (cspC and cspD) were found to be under the negative control of the BvgAS system, the main transcriptional regulator of Bordetella virulence genes. Moreover, a negative effect of slight overexpression of CspB, but not of the other CSPs, on the transcription of the adenylate cyclase toxin CyaA of Bordetella pertussis was observed, suggesting cross-talk between the CSP-mediated stress response stimulon and the Bordetella virulence regulon.