The German Mouse Clinic: a platform for systemic phenotype analysis of mouse models.

The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line--a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, host-pathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.

Local mechanical stimulation induces components of the pathogen defense response in parsley.

Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response to Phytophthora sojae. In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli. Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes. When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected. A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation. By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations. Neither papilla formation nor hypersensitive cell death occurred under the conditions tested. These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes. As yet unidentified signals may be involved in papilla formation and hypersensitive cell death.

Correlation of Rapid Cell Death with Metabolic Changes in Fungus-Infected, Cultured Parsley Cells.

To study in detail the hypersensitive reaction, one of the major defense responses of plants against microbial infection, we used a model system of reduced complexity with cultured parsley (Petroselinum crispum) cells infected with the phytopathogenic fungus Phytophthora infestans. Experimental conditions were established to maintain maximal viability of the cultured cells during co-cultivation with fungal germlings, and a large proportion of the infected parsley cells responded to fungal infection with rapid cell death, thereby exhibiting major features of the hypersensitive reaction in whole-plant-pathogen interactions. Rapid cell death clearly correlated with termination of further growth and development of the fungal pathogen. Thus, the system fulfilled important prerequisites for investigating cell-death-related metabolic changes in individual infected cells. Using cytochemical methods, we monitored the increase of mitochondrial activity in single infected cells and the intracellular accumulation of reactive oxygen species prior to the occurrence of rapid cell death. We obtained strong correlative evidence for the involvement of these intracellularly accumulating reactive oxygen species in membrane damage and in the resulting abrupt collapse of the cell.

Suspensions of plant cells in microgravity.

Electrofusion of evacuolated with vacuolated mesophyll protoplasts of Nicotiana spec. was performed as part of the German Sounding Rocket Program (TEXUS). The results indicate a significant increase not only in the yield of 1:1 fusion products, but also in homo- and multifusion products. Heterokaryons obtained under microgravity have been shown to be viable to a higher degree with respect to their ability for light-dependent oxygen evolution (independent of other substrates than bicarbonate). Furthermore we were able to regenerate hybrid plants from suspensions of vacuolated and evacuolated protoplasts which were pulse-treated under microgravity. These expressed characteristics intermediate to those of the parental plants (Nicotiana tabacum (cv. Samsun), evacuolated; N. rustica, vacuolated).

Determination of physical membrane properties of plant cell protoplasts via the electrofusion technique: prediction of optimal fusion yields and protoplast viability.

By variation of physical parameters (field strength, pulse duration) which result in electrofusion and electroporation, properties of the plasma membrane of different types of plant cell protoplasts were analyzed. The lower threshold for that field pulse intensity at which membrane breakdown occurred (recorded as fusion event) depended on pulse duration, protoplast size, and protoplast type (tobacco, oat; vacuolated, evacuolated). This fusion characteristic of plant protoplasts can also be taken as a measure of the charging process of the membrane and allows thus a non-invasive determination of the time constant and the specific membrane capacitance. Although the fusion yield was comparable at pulse duration/field strength couples of, e.g., 10 µs/1.5 kV*cm(-1) and 200 µs/0.5 kV*cm(-1), hybrid viability was not. Rates of cell wall regeneration and cell division of tobacco mesophyll protoplasts were not affected but may have been increased at short pulse duration/high field strength. Plating efficiency, in contrast, was significantly decreased with longer pulse duration at low field strengths.

Effects of microgravitation on electrofusion of plant cell protoplasts.

Electrofusion of evacuolated with vacuolated mesophyll protoplasts of Nicotiana tabacum was performed as part of the German Sounding Rocket Program (TEXUS 17, 1988). The results indicate a significant increase not only in the yield of 1:1 hybrids, but also in homo- and multifusion products. Hybrids obtained under microgravity have been shown to be viable to a higher degree with respect to their ability for light-dependent O2-evolution (independent of other substrates than bicarbonate). This finding is of interest for fusion experiments where only limited numbers of fusion partners are available (e.g. protoplasts from embryogenic tissues) or where fusion yields are extremely low under 1 x gravity (e.g. protoplasts of different specific density).

Mass electrofusion and mass selection of functional hybrids from vacuolate × evacuolate protoplasts.

Vacuolate mesophyll protoplasts of Nicotiana tabacum L. (cv. Samsun) were electrically fused with evacuolate protoplasts of the same species. For this purpose a mass fusion chamber was constructed. Due to distinct differences in the specific density of the respective protoplast populations, interspecific hybrids could be separated from intraspecific ones as well as from unfused parental protoplasts on an isoosmotic density gradient. The interspecific hybrids appeared to be viable to about 60% as assayed by a bacterial test for photosynthetic oxygen evolution.