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, then a strain 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 here at The Jackson Laboratory . 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; each is described in the text 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 routinely find the abnormal mouse with overt anatomical or behavioral abnormalities. , and such mice are sent to the These mice are set aside, along with their parents and siblings, and sent to the Deviant Search Program. , This search is done usually which occurs in a central location at the Lab twice a month. Once there, 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 here.Test for heritability
Not all phenomutations deviants 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 get use an unrelated mouse from the same or closely related inbred strain background to mate. If the mutant phenotype appears in the offspring than we have proven it is a heritable mutation. Heritability is established if we identify affected offspring from this mating or from a subsequent mating of the first generation (F1) offspring of this mating.Mode of inheritance
We need to determine if whether the heritable mutation displays 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. One reason to use control mice not from the colony is because we know the genotype to be a true wild type. If we mate to a control in the colony, we do not know if that mouse is a carrier for the mutation at this point. And we use controls from a background that is closely related evolutionary so to reduce our chances of modifying genes from a different inbred background that may alter the phenotypic outcome.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. If mutant animals appear in the first generation (F1), than 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, then 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, which is apparent in the heritability crosses described above 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 ones 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. If a strain for allele testing isn’t obvious, we do perform a database search to see determine if there are existing strains unfamiliar to our staff with phenotypes that resemble our new mutant. Mouse Genome Informatics (MGI) is a good web site to start a phenotype search has a wealth of phenotypic data for both spontaneous and targeted mutations to help identify possible candidates.Mapping
Mutations resulting in unique phenotypes are genetically mapped to establish the a chromosomal location of the causative gene. A complete protocol for mapping mutant phenotypes can be found on the web site for the Mouse Mutant Resource, a fellow member of Genetic Resource Science (GRS) here at Jax. Briefly, a mapping cross is established to produce affected mice, either F2s from an intercross or BC1s from a backcross, depending on the mode of inheritance of the deviant trait. We generally use mate our mutant tom.m.CCastaneus (CAST/EiJ) for the mapping cross because of its high degree of genetic diversity and the availability of a large number of polymorphic markers. , a member of the Mus musculus species. This strain has the most polymorphisms from other inbred strains. This allows for us to choose from more primers. However, the same genetic diversity that enables mapping, will often affect the penetrance and presentation of the mutant phenotype when Castaneus may not workCAST/EiJ is used. The mutation may not appear with Casteneus in the bIf this happensackground, so we systematically outcross to other inbred strains in descending order of genetic relatedness until we recapture the mutant phenotype in the mapping cross, balancing the need for genetic diversity for mapping with presentation of the phenotype for identification of affected offspring.
We still try for strains that are genetically different enough to have ample polymorphisms to map. Spleen and tail tips are collected from affected offspring and genomic DNA is prepared for mapping. Once about twenty of these tissue samples have been saved, we extract the DNA and begin a genome scan we initiate a genome scan when approximately twenty affected animals are collected. Regions of the mouse genome are prioritized based upon potential candidate genes, and if these prove to be unlinked, a systematic genome wide scan is performed on pooled DNA. When a potential linked region is identified (recombination frequency of 30% or less), individual DNA samples are tested for the DNA markers in that region. Even if we cannot allele test, we still look for similar phenotypes and where they map. If we find some candidate genes, we run our DNA at these loci. If we do not find our mutation this way then it is time to systematically map through the genome until we find the map location. For dominant mutations, we test individual DNA with three or four MIT makers that span each chromosome. If the entire pooled DNA looks to all or mostly amplify the mutant DNA band and not the out-crossed strain band, then we run individual markers at those loci. Linkage is confirmed by testing additional markers in the area to narrow the critical region containing the mutation. Thirty percent can mean random recombination the same as 50% or it can mean we are closing in on the critical region. If the recombination continues to decline as we move more proximal or distal, we know we are close to a chromosomal location. Zero percent recombination with a distal and proximal flank is our goal.At this point, the mapped location of the new mutant is presented on the Craniofacial Mutant Resource web site and a descriptive narrative is prepared for web publication. For a few mutations, we proceed to gene identification using positional cloning techniques.
Candidate genes
We try to narrow our critical region in order to examine a manageable list of candidate genes. MGI and Ensembl are good web sites for this search. Sometimes there is a gene in our region that resembles our phenotype. If our strain is recessive, we can try and obtain the strain and do an allele test. At this point we decide if we have done enough research on the strain or if we want to try and clone the gene. Most strains will go public at this juncture and be available for outside investigators to continue the research. A determining factor to make a strain public or to pursue for our own research is how many strains with this gene mutation are available. Or is our mutation novel? An MGI search can quickly answer the question. If we have decided we want to continue to invest our time and money, we can start sequencing the candidate gene.
Phenotypic analyses
Hearing
Mutant and control mice are assessed for hearing by Auditory Brainstem Response (ABR) by our collaborators in theVision
Our collaborators in the GRS Ocular 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 is 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 found 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 achieved 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
For Many mutant strains, will get a chromosomal location and undergo our standard biological characterization the causative mutation is mapped to a modest (5-10 cM) interval and basic biological characterization is done (described above). If we decide that is adequate, the paper goes onto our Craniofacial web site and the strain is available for public distribution. These data are provided online and are linked from the “Mapped Mutants” page of the Craniofacial Mutant Resource.
