2012 Standard Protocols and Procedures
Mouse Colony Maintenance
Craniofacial Resource mice are housed in 51 square inch polycarbonate boxes, on bedding composed of sterilized shavings of Northern White Pine, under 14:10 hour light:dark cycles. A diet of autoclaved NIH 31 (6% fat diet, Ca:P of 1.15:0.85, 19% protein, vitamin and mineral fortified; Purina Mills International, Richmond, IN) and water acidified with HCl to achieve a pH of 2.8-3.2 (which prevents bacterial growth) are freely available. Mouse colony maintenance and use is reviewed and approved by The Jackson Laboratory Institutional Animal Care and Use Committee and is in accordance with The National Institutes of Health guidelines for the care and use of animals in research.
PIXImus Densitometry
PIXImus scans (PIXImus, LUNAR, Madison, WI) which provide skeletal and body composition data such as Bone Mineral Density (BMD, g/cm2), Bone Mineral Content (BMC, g/cm2), body mass (g), lean mass (g), fat mass (g), and % fat mass, are completed on groups of 6 male and 6 female 12 week old mutant and control mice. The skulls and bodies are scanned separately to provide independent data on skull BMD and BMC and body BMD and BMC. The PIXImus small animal densitometer (DEXA) has a resolution of 0.18 x 0.18 mm pixels and is equipped with software version 1.46 . The PIXImus is calibrated routinely with a phantom utilizing known values, and a quality assurance test is performed daily. The variability in precision for measuring total body BMD is, less than 1%, and approximately 1.5% for specialized regions such as the skull. The correlation between PIXImus BMD measurements of 614 lumbar vertebrae compared to peripheral quantitative computerized tomography (pQCT) measurements was found to be significant (p<0.001; r=.704) (Donahue, 1999) .
Faxitron X-rays
X-rays at 5X magnification of the skull and at 3X magnification of the body of a male and female mutant and control at 12 weeks of age are obtained using a Faxitron MX20 cabinet X-ray (Faxitron X-Ray Corp., Wheeling, IL. USA) and Kodak Min-R 2000 mammography film (Eastman Kodak Co., Windsor, CO, USA). X-rays are then analyzed to determine the specificity of the skeletal phenotype.
Skull Preparation
Skulls of 6 male and 6 female mutants and controls are collected at 12 weeks of age, prepared by incomplete maceration in potassium hydroxide, stained with alizarin red, and stored in undiluted glycerin (Green, 1952). During the collection process, right ear pinnae are measured with digital hand calipers (Stoelting, Wood Dale, IL, USA).
Hand Caliper Skull Measurements
Seven measurements taken with hand held digital calipers are used routinely to define skull morphology at the Lab's craniofacial resource. These measures have a high degree of accuracy and precision in our hands and are able to discriminate differences between mutant and control skull characteristics. Our linear measures have been added to the illustration below which was taken from a publication by Dr. Joan Richtsmeier characterizing craniofacial differences in mouse models of Down Syndrome using three dimensional anatomical landmarks (Richtsmeier, 2000). Skulls are cleared with potassium hydroxide and stained with alizarin red dye in preparation for caliper measurements to be taken.
Skeletal Preps
In many cases whole skeletons of mutant and control mice are cleared in 1% KOH, stained with alizarin, stored in glycerin (Green, 1952) and then evaluated for skeletal malformations. Malformations found can indicate that the craniofacial phenotype is part of a greater syndrome.
Data Analysis
Hand caliper skull measurements and PIXImus skeletal and body composition data are evaluated using StatView 4.5 software (Abaccus Cary, NC) for Macintosh computers. Differences are considered significant when p < 0.05.
Molecular 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 (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.
Sequencing
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 (Fairfield, et al., 2011). 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.
References
Donahue LR, Rosen CJ, Beamer WG. (1999) Comparison of Bone Mineral Content and Bone Mineral Density in C57BL/6J and C3H/HeJ Female Mice by pQCR (Stratec XCT 960M) and DEXA (PIXImus). Thirteenth International Mouse Genome Conference. Philadelphia, PA.
Fairfield H, Gilbert GJ, Barter M, Corrigan RR, Curtain M, Ding Y, D'Ascenzo M, Gerhardt DJ, He C, Huang W, Richmond T, Rowe L, Probst FJ, Bergstrom DE, Murray SA, Bult C, Richardson J, Kile BT, Gut I, Hager J, Sigurdsson S, Mauceli E, Di Palma F, Lindblad-Toh K, Cunningham ML, Cox TC, Justice MJ, Spector MS, Lowe SW, Albert T, Donahue LR, Jeddeloh J, Shendure J, Reinholdt LG. 2011. Mutation discovery in mice by whole exome sequencing. Genome Biology 12: R86.
Green, MC. (1952) A rapid method for clearing and staining specimens for the demonstration of bone. The Ohio Journal of Scince 52(1):31-33. January 1952.
Manly KF, Cudmore RH, Jr., Meer JM. (2001) Map Manager QTX, cross-platform software for genetic mapping. Mamm. Genome 12:930-932.
Richtsmeier JT, Baxter, LL, Reeves, RH. (2000) Parallels of craniofacial maldevelopment in Down syndrome and Ts65Dn mice. Dev. Dyn. Feb;217(2):137-45.
Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML. (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and Tris (HoSHOT). Biotechniques 29;52-54.
