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Director
William Philbrick, Ph.D., is an Associate Professor of Medicine and Director of the Molecular Core. He is a molecular and cellular biologist with extensive experience in gene expression systems both in cultured cells and in animal models. Bill has been actively involved in training others in the use of the methodologies offered by the Core for the past 13 years. He has served as the Director of the Molecular Core of the Yale Diabetes Endocrine Research Center since 1994. He has carried out a series of experiments directing overexpression of parathyroid hormone-related protein (PTHrP) to endogenous sites of expression in transgenic mice which have implicated PTHrP as a developmental regulatory molecule.
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General Overview
The mission of the Molecular Core is 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.
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Core Services
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- 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.
- Real-Time PCR
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.
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Research
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Inducible transgene strategy.
We typically employ splice/polyadenylation/termination sites from the SV40 small t gene for the inducer transgene and from human growth hormone for the responder transgene.
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Primers and RNase protection probes for tetracycline transactivator and growth hormone sequences allow for rapid identification of transgenic animals and relative quantitation of mRNA expression levels, respectively. A number of bone- and/or cartilage-specific promoters and both tetracycline-inducible and tetracycline-repressible transactivators are available.
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Gene targeting strategies.
A) Schematic of a ß-galactosidase knock-in construct. This approach utilizes the standard design of a knock-out construct (including appropriate lengths of genomic homology, and neomycin and thymidine kinase cassettes for positive and negative selection, respectively), but also includes a cassette incorporating an artificial splice acceptor, a picornaviral internal ribosomal entry site and stop codons in all three reading frames followed by a ß-galactosidase coding region with its own translational initiation codon and polyadenylation/termination sequences. B) Schematic of a conditional knock-out construct. Modifications to the standard design include frt sequences flanking the neomycin cassette which will mediate its recominational excision on induction with the Flp recombinase either by transient transfection of ES cells or through the use of a transgene in vivo. Transgenic expression of Cre recombinase then excises a critical exon in the targeted gene in a tissue-specific manner.
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Last Edited
July 13,2001 TD
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Localization of gene expression.
In situ hybridization of normal mandible sectioned sagittally through the molar crypt and hybridized with murine probes for the PTH/PTHrP receptor (A) and PTHrP (B). The receptor is found principally in bone (b), but can also be detected in the dental mesenchyme, both in the dental pulp (dp) and the dental follicle layer (df).
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PTHrP localizes to the outer enamel epithelium (oe) and the stellate reticulum (sr). Some enamel (e) remains after decalcification; d, dentin. The scale bar represents 0.12 mm.
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