Development of new mouse models with craniofacial abnormalities
Newly discovered phenodeviants with craniofacial abnormalities are evaluated for their potential value as new models using a standard set of genetic and phenotypic tests. If the craniofacial dysmorphology is heritable and consistent, a colony carrying this phenotype is established, and the new model is made available to the scientific public either via a peer reviewed publication or on our craniofacial web site. The following overview describes our process and offers a model for others who wish to characterize craniofacial mutations in their own laboratories.
We proceed in a series of steps described in detail below:
Deviant search
As part of The Jackson Laboratory quality assurance program for its production colonies, Animal Care Technicians are trained to observe numerous mice within an inbred strain, and routinely find abnormal mice with overt anatomical or behavioral abnormalities. These mice are set aside, along with their parents and siblings, and sent to the Deviant Search Program. Mice are evaluated as to the uniqueness of their abnormality and are offered to any staff member who is interested in working on the new deviant. Members of the Craniofacial Resource routinely attend Deviant Search, and the majority of new models we develop come from this program; other deviants are offered to us from individual research colonies.Test for heritability
Not all phenomutations pass their unique trait on to subsequent generations. We determine heritability by mating the affected mouse to an unaffected parent or sibling. If a relative is not available, we use an unrelated mouse from the same, or closely related, inbred strain background as a mate. If the mutant phenotype appears in the offspring from this mating, or from a subsequent mating of the first generation (F1) offspring, the mutation is heritable.Mode of inheritance
To determine whether the heritable mutation follows a dominant or recessive inheritance pattern, we mate the affected mutant mouse to an unrelated wild type (unaffected) mouse that has the same or similar inbred background. It is important to use unrelated mice for this testing because the genotype of siblings is not known, and a clinically normal mouse could be a carrier, complicating the interpretation of the heritability test. We use a control from a background that is closely related evolutionary to reduce our chances of introducing modifying genes from a different inbred background that may alter the phenotypic outcome. We have found that most phenotypes are more penetrant on the background in which they arose than on other strains, indicating the presence of one or more modifying genes interacting with the causative gene.
If mutant animals appear in the first generation (F1), we can conclude that the new mutation is dominant or semi-dominant. If mutant animals appear in the second generation (F2), and not in the first, then the mutation has a recessive mode of inheritance. If the appearance of the F1 and F2 affected mice differs, the mutation is semi-dominant. As a rule of thumb, we generally screen twenty F1 and twenty F2 offspring.
Penetrance
Heritability is not enough. Many craniofacial mutants display incomplete phenotypic penetrance where the severity of the phenotype varies in affected mice. Heritable phenotypes with low penetrance are significantly more difficult and costly to maintain and map, and we typically do not pursue strain development when the penetrance of the phenotype is less than 20%. The mutation has to express itself enough in a colony to justify the time and expense. While Mendelian numbers are ideal, we may keep a colony that produces mutants down to 20% of the population. Ten percent or less expression usually results in terminating the research of a colony.Genetic analysis
Remutations
Many newly discovered deviant phenotypes resemble mutations that have been previously identified. When this happens, a complementation or allele test is done right away to test for a remutation to a known gene. We may also utilize the Mouse Genome Informatics (MGI) database to help search for established strains with known genes that may have a similar phenotype to our mutation. If an established strain is available, we can order a mouse to do a complementation test.Mapping
Mutations resulting in unique phenotypes are genetically mapped to establish the chromosomal location of the causative gene. In the past, the Craniofacial Mutant Resource relied on high-resolution mapping to narrow the genetic interval to a manageable size for candidate gene analysis (see protocols used prior to 2012). With the advent of sequence capture and high throughput sequencing (HTPS) techniques, this level of resolution is no longer required. Genetic mapping to rough chromosomal position still provides a number of useful advantages however, such as a reduced computational burden, fewer variants to validate, and greater confidence in variant causality. To identify causative mutations we use a combination of genetic mapping and HTPS. We have found that mapping a gene at least to a chromosome greatly facilitates the analyses of HTPS. Our studies use a combination of 1-10 Mb interval-specific, gene-specific, and whole exome approaches to identify a wide spectrum of mutation types across diverse genetic backgrounds.
Exome capture and sequencing
Once linkage is established and a broad chromosomal location identified, we employ whole exome capture and HTPS to identify potential causative variants. Whole DNA exomes from mutant samples are captured using an in-solution, hybridization-based probe pool developed in our group in collaboration with Roche-Nimblegen. The content of the probe pool is defined by the unified mouse gene catalog which, excluding UTR sequences, olfactory receptors and pseudogenes, encompasses approximately 50 Mb of genomic sequence. Our preliminary exome data indicate high capture sensitivity and specificity, >96.7% of the targeted bases covered with just one lane of 75 bp paired-end on the Illumina GAIIx.
Our primary sequencing approach is to sequence whole exomes from enriched mutant DNA samples and to multiplex where possible. An additional advantage of the paired end sequencing approach is that it provides positional information that is critical for the identification of spontaneous mutations that are due to genome rearrangements (larger insertions or deletions).
Analysis and validation
All raw sequence data analysis, including read mapping and SNP/mutation calling are performed by the Computational Science service at JAX, using Galaxy sequence analysis tools. Multiple candidate variations are detected in each strain, but most are eliminated upon validation. For validation, each candidate mutation is PCR amplified from up to 10 other individuals within the same mutant pedigree. Each PCR amplimer is subjected to Sanger sequencing. In the majority of the cases, non-mutagenic variants will not segregate with the phenotype, but bona fide mutations will. Validation is not attempted until sufficient sequencing coverage has been obtained, as indicated by comparison of computational analysis of parameters like '% target bases covered' and by comparison of the variant profiles obtained to the Sanger whole genome sequencing data.
Phenotypic analyses
Hearing
Mutant and control mice are assessed for hearing by Auditory Brainstem Response (ABR) by our collaborators in the Hereditary Hearing Impairment program.Vision
Our collaborators in the Eye Mutant Resource screen mutant and control mice for eye abnormalities first by clinical examination. If an irregular eye phenotype is suspected, further screening procedures are done by slit lamp examination or ERG.Pathology
One mutant and one control mouse are examined for anatomical lesions by our GRS collaborating pathologist, Dr. Roderick Bronson. If a lesion is found, more mice and appropriate sectioning and staining are done to confirm the histopathological finding.
Skeletal morphometry and bone density
The axial and appendicular skeleton are examined for abnormalities that might accompany the craniofacial phenotype. X-rays (Faxitron X-Ray Corp., Wheeling, IL) and densitometric measurements by PIXImus (PIXImus™, Fitchburg, WI) provide skeletal structure and bone mineral density information. Three-month-old mutants and controls from both sexes are analyzed; see details.Craniofacial measurements
Hand held digital calipers are used to measure skull morphology. Three-month-old mutants and controls from both sexes are analyzed using standard anatomical landmarks.Developmental phenotyping
Strains with dominant craniofacial mutations are tested to determine if a more severe phenotype, possibly embryonic lethal, results when the mutation is made homozygous. Heterozygous mice are intercrossed and litters are scored for expected number of pups, clinical appearance, and typed for markers at the established interval. If no homozygotes are found, timed matings between F1 animals are set up, and embryos are harvested at E18.5, E14.5 and E10.5. If homozygotes are not identified, the investigation is no longer pursued. If validated homozygotes are found, they are examined for phenotypic abnormalities and the results are presented in web paper format (described below).Strain preservation
Cryopreservation
Most strains are archived via frozen sperm; others require cryopreservation of embryos, depending on the type of mutation and the genetic background of the mutant strain.
DNA
DNA is saved in the DNA Resource at JAX.
Publications
Web Papers
Once a causative mutation is mapped to a gene or a modest (5-10 cM) interval and basic biological characterization is done (described above). If we decide that is adequate, we will publish the results in a paper on our Craniofacial web site and the strain becomes available for public distribution. These data are provided online and are linked from the “Mouse Models” page of the Craniofacial Mutant Resource.
