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 TJL'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
Molecular mapping is completed using DNA extracted from tail or spleen through a Hot Sodium and Tris (HotSHOT) protocol (Truett, 2000). Primer pairs (MapPairs, Research Genetics, Huntsville Ala.) of microsatellite markers are used to establish and refine the initial mapping location. PCR products are visualized and scored via gel electrophoresis and ethidium bromide staining. Linkage analysis and recombination frequencies are calculated via the Map Manager computer program (Manly, 2001) and compared to known and predicted gene location data and marker location data published in the Celera (www.celera.com/) and Ensemble (www.ensembl.org/Mus_musculus/) genome databases and the Mouse Genome Informatics website (MGI, www.informatics.jax.org/).
Sequencing
Primers are designed using published cDNA sequences from the Ensemble or Celera mouse genome databases. DNA is amplified through PCR and separated via gel electrophoresis. The resultant products are then removed and purified with QIAquick Gel Extraction Kits (Qiagen, Inc., Valencia, CA, USA) and sequenced using an Applied Biosystems 373A DNA Sequencer and an optimized DyeDeoxy Terminator Cycle Sequencing method.
References
Donahue, L.R., Rosen, C.J., Beamer, W.G. (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.
Green, M.C. (1952) A rapid method for clearing and staining specimens for the demonstration of bone. The Ohio Journal of Science 52(1):31-33. January 1952
Manly, K.F., Cudmore, R.H. Jr., Meer, J.M. (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, G.E., Heeger, P., Mynatt, R.L., Truett, A.A., Walker, J.A., Warman, M.L. (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and Tris (HoSHOT). Biotechniques 29;52-54.
