Scientific Overview
The mouse has emerged as the premiere experimental mammal because of the ability to analyze known genes and for the variety of new heritable phenotypic variants available for study. Mouse genetics has made significant contributions to several complex areas of mammalian biology, but the central nervous system (CNS) represents a challenge because of its massive complexity and great degree of plasticity.
Recent progress in gene cloning, expression and gene targeting promise to further our knowledge, but genotype-based approaches alone are insufficient due to the complexity and redundancy of CNS functions. The next step is to systematically collect a large number of mouse mutations for specific neurological disorders and mechanisms. Large-scale mutagenesis offers a progressive resource towards this end.
The Jackson Laboratory has established a Neuroscience Mutagenesis Facility to produce new mouse models of human neurological disease. The facility will generate new mutations using N-ethyl-N-nitrosourea (ENU) in both genome-wide and specific genomic region screens, in both normal C57BL/6J mice and sensitized mutant mice. This classic strategy will be complemented by implementation of new mutagenesis technologies utilizing embryonic stem cells and other mutagens to increase efficiency.
Overview of Genetic and Phenotyping Strategies
Mutagenesis and phenotypic screens offer a powerful way to identify genes not previously known to participate in neurological processes. However, because of the great variation in susceptibility of individual loci to mutation, and differences in the overtness of any consequent phenotype, saturation mutagenesis is not the primary goal of our program. Instead, our view is that screens are valuable for identifying genes with a sufficient level of unique function that they can serve as entry points into pathways.
Choice of mutagen. We will initially use ethyl nitro-sourea (ENU) because it is the most efficient mutagen currently available. However, because it is far from ideal, we are also developing the use of other mutagens and alternative breeding strategies (ES cells), as well as the use of chromosome deletions and markers to improve mutagenesis.
Choice of strain. C57BL/6J (B6) is our strain of choice for genome-wide screens and genome sequencing efforts, because so many existing mutations either occurred or have been bred to this background, facilitating phenotypic comparison. However, a smaller percentage of mice screened are of A.B6-Tyr+ origin (25%), while others (25%) are of mixed background (C57BL/6J x BALB/cByJ) or are derived from mutagenized embryonic stem cells.
Dominant vs. recessive. There has been much discussion in the community on whether to screen for dominant or recessive mutations. An advantage of screening for dominants is that they are available on a genome-wide basis in one generation, while recessives require three. However, it is uncertain what classes of genes can give dominant mutations; it is unlikely that all will. Additionally, estimates suggest the frequency of recessives is four- to ten-fold higher. (The experience in TJL production colonies suggests that for visible mutations the difference could be 25 times higher). Since all of the present large-scale mutagenesis efforts have screened for dominants, our program emphasizes recessives, recognizing that dominants will also be obtained. This strategy will also provide general information on the relative frequency of the two types of mutations.
Mapping. The NMF will undertake reaonable efforts to determine the chromosomal regions of the mutations, and whenever possible identify allelic relationships to known genes by complementation analysis (see overview) ; however, detailed mapping of the mutation is beyond the scope of the program, and left to the individual investigators who purchase these mutants.
Phenotyping Scope. A major challenge in phenotyping is weighing the need to gather sufficient information on each mutation against the need to screen large numbers of animals to gain broad coverage of the genome. Proper balance of these factors will maximize the eventual recovery of interesting new mutations. Our approach has been to devise efficient screens for a series of phenotypic domains, favoring high-throughput, simple tests (and multiple tests where possible), and then to rely on more sophisticated confirmatory testing of suspect mice. Our choices of phenotypes are based on the expertise of JAX staff and collaborators, in consultation with our External Advisory Board, and presently, pedigrees are screened for abnormalities of motor function, epilepsy, vision, hearing, gustation, neuro-developmental as well as a multi-domain screen using a Comprehensive Lab Animal Monitoring System (CLAMS™). In addition, mice of mixed background, C57BL/6J and BALB/cByJ are placed in an aging colony to be screened for age-related disorders. Standard histology is performed on all mouse lines selected for further studies.
Links to protocols: Observation; CLAMSTM (former CCMS); Gait Analysis; Gustation; Vision and eye; Seizure threshold; Creatine kinase; Auditory brainstem response (ABR); Acoustic startle response (ASR): Developmental Screen.
