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. 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.
Gene targeting strategies

Gene targeting strategies

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.

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). 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.