institute of experimental genetics (ieg)

mutants generated

1. Objectives

With respect to the genetics and pathogenesis of human diseases, animal models are indispensable for further investigations. For this purpose, the Munich ENU Mouse Mutagenesis Screen was founded in 1997 and serves as a platform for systematic, genome-wide production and analysis of mouse mutants as a model system for inherited human diseases. In the Munich ENU Project (MEP) mouse mutants are produced systematically in a large scale by chemical mutagenesis using the alkylating agent N-ethylnitrosourea (ENU).

2. ENU mutagenesis

The mouse is intensively used as a model system due to the similarity of genome organisation, developmental and biochemical pathways and physiology. Since most of the functional properties of biological molecules are still unpredictable from pure sequence analysis, an extensive gap between sequence information and knowledge about their function in the organism has to be filled. Different approaches have been used in the last years to create informative mouse models, known as forward (phenotype-driven) genetics. ENU is currently the most powerful mutagen for the production of mutants in mice (Russell et al. 1979, Russell et al. 1982, Peters 1985, Dove 1987, Russell et al. 1990, Favor et al. 1990, Rinchik 1991). The mutations recovered after ENU-mutagenesis are mainly point mutations, i.e. A-T base pair substitutions and/or small intragenic lesions (Popp et al. 1983, Harbach et al. 1992). Many of the mutants produced by ENU will therefore be hypomorph (partial loss-of-function), although gain-of-function as well as complete loss of function mutants can also be expected (Grunwald and Streisinger 1992, Justice and Bode 1988). Therefore, the availability of point mutations is very important for a more detailed functional analysis of many genes.

ENU shows mutagenic action on premeiotic spermatogonial stem cells (Russell et al. 1979, Rinchik 1991). This allows the production of a large number of F1 founder animals from a single treated male, minimizing the number of animals required and the handling of ENU. Mutations induced by ENU will not be tagged molecularly , as it is the case in reverse genetics, with gene trap or transgenic insertional mutants. Although this is initially a disadvantage with respect to the cloning of the responsible genes, and have to be analysed by linkage analysis and positional cloning. the availability of point mutations is very important for a more detailed functional analysis of many genes.

3. The Munich ENU Project

The central part of the ENU mouse mutagenesis screen is the Core Facility for coordination and management of the complete workflow. The Core Facility is the platform for mouse breeding, mutagenesis of males, sample taking and shipment of blood and tissue samples from offspring of mutagen treated C3HeB/FeJ mice to all the associated research groups participating in the mutant screen. For some screens, measurement of blood samples can be performed. Animal management and sample tracking are supported by a database (MouseNet) developed at the Institute of Experimental Genetics.

Structure of the ENU Screen

In the Munich ENU project , male C3HeB/FeJ mice are treated with three ENU injections in weekly intervals. After a sterility period, injected males are mated to wildtype untreated females. Only F1 litters deriving from ENU males with a minimal sterility period of 80 days are chosen for phenotyping to ensure that they were generated with mutagenic treatment derived sperm. A screening and phenotyping protocol for pathophysiological abnormalities – the Munich protocol – has been established in the last years to assess mutant phenotypes for specific, postnatal abnormalities comprising congenital malformations, clinical chemical, biochemical, immunological defects and complex traits like allergy and behaviour. According to this standardised protocol a total of 135 parameters can be evaluated. First phenotyping for dysmorphological malformations is done at weaning time of the litters at the age of three weeks. F1 mice are preserved for breeding the potential mutation. The screen for recessive mutations involves two generations of breeding. Mutant mouse lines obtained by the recessive trait are subjected to outcross/backcross breeding mapping followed by linkage analysis using SNPs (single nucleotide polymorphisms) at our institute.

Initially, two different strategies were applied in order to screen for either dominant or for recessive mutants. In both cases, male mice are injected with ENU and then mated to females in order to produce F1 founders. These F1 mice can either be analysed directly for dominant mutations or bred further to subsequently study recessive phenotypes. Very large numbers of mice can be analysed in a dominant F1 screen. In this case, all F1 mice are screened for phenotypic abnormalities. Screening for recessive mutations involves two generations of breeding. From F1 founder males, F2 female offspring are raised, half of which are heterozygous for the newly induced mutations. Backcrossing F2 to the F1-founder male or intercrossing the F2 is then carried out to identify recessive mutant phenotypes among the F3 offspring.

From the breeding scheme, it is evident that a recessive screen is more time- and space consuming compared to the dominant breeding strategy (figure 1). Therefore, the recessive breeding scheme has been stopped end of 2004 for genome-wide mutagenesis screen.

Breeding scheme of the ENU screen

Aim is the annual production, phenotyping and archiving of 1600 F1 males, additionally, 1600 female F1 animals are phenotyped for dysmorphologies and associated blood screens. Main focus of the Dysmorphology screen is on bone and cartilage phenotypes. Large scale DEXA scan examination of F1 animals is therefore a promising tool for the research on aetiologic reasons for osteoporosis.

New combined workflow of the random (MEP) and the sensitised (SENS) mutagenesis screen

The parameters analysed in our screen comprise for none blood-based parameters body: body size, body shape, right-left-abnormality, skull shape, whiskers, tooth lengt, thooth shape, tooth colour, ear size, ear form, eye size, cataracts, limbs, double digits, crippled digits, syndactylism, nails, kinked tail, coiled tail; physical appearence: general physical condition, weight, coat colour, coat structure, skin colour, skin structure, swellings, carcinoma/ tumors, strength, gait, trembling, cramping, paralysis, seizures, respiration; behaviour: general behaviour, activity, agressiveness, exploring, head tossing, head shaking, circling, click-box-test, landing behaviour, hanging behaviour, articulation; environment: Social structure in cage, cage cleanness, urine, faeces; additional X-ray and bone densiometry.

Blood-based parameters :

Clinical chemical parameters: 39 tested in established lines

• Basic hematology: WBC, RBC, HCT, MCV, MCH, MCHC, PLT
• Differential blood count: SEG, LYM, MOZ, EOS, BAS
• Plasma enzymes activities: AP, AMY, CK-MB, GOT , GPT , LIP, LDH
• Plasma concentrations of specific metabolites: GLU ,TP, uric acid , UREA , CREA, TRF, FER, BILI, HDL, LDL, TG, CHO, Fe , unsaturated iron binding capacity (UIBC), ALB
• Plasma concentrations of electrolytes: K , Na , Cl , Ca , inorganic P

F1 Screen only: urea, uric acid, CREA, TP, GLU

Haemostasis (3)

• Thrombelastography: fibrin formation rate, clot growth rate, fibrinolysis
• Biochemical metabolites: steroid metabolites

Immunological parameters (24):

• basal immunoglobulin levels: IgM, IgG1, IgG2a, IgG2b, IgG3, IgA;
• Lymphocyte populations and lineage markers: CD3, CD4, CD8, CD19, CD21, CD25, B220, CD5, IgD, DX5, F4/80, CD14, CD11b, Gr-1, gdTCR, CD45RA, MHC class II
• auto-reactive immunantibodies: anti-DNA antibodies

Allergy (1):

• immunglobulin E level

Lysosomal enzymes (4):

• lysosomal sorting enzymes: b-hexosaminidase, b-mannosidase,b-galactosidase ; lysosomal sulfatase

Mitochondrial diseases (2):

• plasma concentration of lactate; morphology of mitochondria by electronmicroscopy

Dysmorphology Screen (4):

• alkaline phosphatase, calcium, phosphorus, osteocalcin

Aldosterone (1):

• aldosterone

Besides this genome-wide mutagenesis project a modifier Screen has been started using Delta-1 (Dll1) knockout mice. Our main interests are the understanding of the Delta/Notch pathway. This signal transduction pathway is well conserved and plays a central role in mediating cell-to-cell communication regulating the determination of various cell-fates during development. In particular, Delta/Notch plays an important role in the cellular interactions underlying somitogenesis, neurogenesis and the development of several organs (Apelqvist et al. 1999, De Bellard et al. 2002, Grandbarbe et al. 2003, Hrabé de Angelis et al. 1997). Moreover, recent studies indicate that the Delta/Notch pathway belongs to a sophisticated system of interactions, which is believed to be involved in a wide range of common diseases including cancer, immunological diseases, infections as well as cardiovascular and neurological diseases. Genetic modifier screens, which have been demonstrated to be very powerful in the fly, are also feasible in the mouse. Combining knockout technology and ENU mutagenesis, we are carrying out a modifier screen with the Dll1 knockout in order to identify modifiers of the Delta/Notch pathway.

C57BL/6J males are injected three times within one week with 80 mg/kg body weight and after 50 days mated to 129Sv isogen R1 heterozygous Dll1^lacZ females. We use the heterozygous mice since the homozygous are embrionically lethal. The F1 animals generated are screened for dysmorphological, clinical chemistry and immunological variations, measuring the parameters indicated previously. The heterozygous Dll1^lacZ animals showing a phenotype are mated to Dll1^lacZ animals and the G2 generation is screened for confirmation of the heredability of the mutation. By this means we have successfully generated 49 new mutant lines. Of major interest for us are the 7 mutant lines that show a Dll1^lacZ-dependent phenotype (Rubio-Aliaga, I. et al., 2007).

Breeding scheme for the Sensitised Screen

Main focus is now on backcross mapping/sequencing and phenotypic characterisation of these mutant mouse lines with a planned annual capacity of 30 outcross/backcrosses. A new technology platform based on SNP markers and MALDI-TOF (Picture1) mass spectrometry was implemented, which enables us to carry out chromosomal mapping in high-throughput manner (Picture2). Mapping is performed with a panel of 158 genomewide SNP markers resulting in 15 mbp intervals. To facilitate the retrieval of appropriate SNP markers and flanking sequences, a tool named "Advanced Retrieval Tool for SNPs" (ARTS) has been developed. By applying several filters, highly reliable SNP marker sequences for several different mouse strains can be extracted from previously imported data-sets like the mouse dbSNP database. More than 60.000 genotypes belonging to independent mouse lines are generated per run. To handle these data, the MyGenotype Maintenance and Analysis System has been developed. Genotype information of analysed mouse lines can be displayed in HTML format along with linkage analysis by c² determination. Alternatively, data can be exported to Map Manager QTX, dChip and R/QTL for further analysis. Further phenotypic characterisation of selected mutant lines can be performed in more detail in the German Mouse Clinic (GMC).