The mission of the Molecular Core is to support the creation and analysis of animal models relevant to musculoskeletal disease and to facilitate the introduction and use of a range of molecular genetic methods by member investigators. To this end, we will provide both services for a number of specialized methodologies and technical assistance in a variety of essential molecular techniques. A central goal is to form an integrated network within the CCMD; the creation of animal models of gene dysregulation and the characterization of target gene expression will be fully supported by the Molecular Core, with further analysis both in vivo and in vitro provided by the Cell and Physiology Cores.
The construction of transgenes and gene disruption vectors
This will include conventional transgenes, inducible transgenes using the tetracycline transactivator system, conventional gene targeting vectors (knock-out and knock-in) and conditional gene targeting vectors using the Cre-loxP and/or Flp-frt methods.
The identification of genetically altered or naturally occurring mutant mice
Genotyping of transgenic and gene-targeted animals will be carried out by PCR on Southern Blot analysis of tail DNA. Other known mutations may be identified by restriction fragment length polymorphism or by PCR-based sequencing.
The localization of gene expression by in situ hybridization
This will include the dissection and fixation of tissues, embedding in paraffin or OCT, sectioning by microtome or cryostat, preparation of riboprobes or oligoprobes, hybridization, emulsion autoradiography and image analysis.
Used for the detection and quantitation of mRNA for experiments requiring greater sensitivity than RNAse protection assays. This may also be used for allelic discrimination and determination of gene copy number.
The quantitation of gene expression
RNase protection provides a sensitive means of either relative or absolute quantitation of mRNA. Normalization is achieved by simultaneous evaluation of endogenous markers such as actins, cyclophilins or GAPDH. Sense controls in the form of unlabelled cRNA standards can be used to provide absolute quantitation when required.
Technical assistance for both basic and advanced methods in molecular biology
This ranges from hands-on training at the bench to consultative functions, such as experimental design, data interpretation and trouble shooting. Supported methods include Southern and Northern blotting; RNA and genomic DNA preparation and analysis; stable or transient cell transfection by calcium phosphate, liposome or electroporation; reporter gene analysis using CAT, luciferase or growth hormone; the construction of vectors for the expression of proteins in bacteria or in eukaryotic cell lines using dominant selectable markers; cDNA and genomic library construction; and competitive PCR, quantitative RT-PCR and PCR-based sequencing.