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Non-primate Lentiviral Vector Administration in the TMJ

¹ Eastman Department of Dentistry, and
² Department of Neurobiology & Anatomy, School of Medicine & Dentistry, University of Rochester, 625 Elmwood Ave., Rochester, NY 14620;

View larger version (79K): [in a new window]   Figure 1. Development of the control FIV('lac) vector with inactive ß-galactosidase gene. (A) The reporter gene lacZ was inactivated after deletion of a critical placZ DNA fragment containing the ß-galactosidase gene transcription initiation site by restriction enzyme-mediated excision and re-ligation of the backbone vector. (B) The structure of mutated FIV('lac) and wild-type FIV(lacZ) viral vectors was confirmed by PCR following transient transfection into the murine cell line NIH 3T3. The presence of viral DNA in cells was detected by a 444-bp DNA band utilizing the "FIV" primers (as depicted in panel A). The complete structure of the lacZ gene was confirmed by a 1.7-kb DNA band utilizing the lacZ primers (depicted as UP, LP in panel A). In the case of the mutated FIV('lac), there was a lack of the 1.7-kb DNA band as the annealing site for the lower primer LP was excised. (C) Deletion of the lacZ transcription initiation sequence in the FIV('lac) resulted in inactivation of the ß-galactosidase reporter gene, as demonstrated by the lack of X-gal staining compared with (D) the FIV(lacZ) vector.

View larger version (61K): [in a new window]   Figure 2. FIV(lacZ) injection (a total of 5 x 106 infectious particles) into the right TMJ resulted in widespread infection of hard as well as soft tissues of the joint. (A) Sagittal TMJ sections analyzed by ß-galactosidase immunohistochemistry and counter-stained by nuclear fast red revealed expression of the reporter gene lacZ in the hypertrophic zone of the condyle, comprised primarily of cartilaginous cells (B), as well as in the meniscus, endothelial cells, and perivascular osteocytes. Panel C depicts TMJ sections from a saline-injected animal. c = condyle; d = disk; m = muscle; v = vessel.

View larger version (57K): [in a new window]   Figure 3. FIV(lacZ) and FIV('lac) injections (5 x 106 infectious particles) in the right TMJ of mice resulted in successful infection of primary sensory neurons located in the ispilateral trigeminal ganglion. The animals’ left-side TMJ was not treated. (A) The presence of backbone FIV DNA in the right trigeminal ganglia ipsilateral to FIV injections was detected by a 444-bp DNA band in lanes 1 and 3, utilizing the "FIV" primers (as depicted in panel A), suggesting successful transduction of the trigeminal sensory neurons by FIV vectors. Lanes 2 and 4 do not display any viral DNA, since they represent left-side ganglia. (B) The inactive form of the ß-galactosidase gene in transduced neurons was detected by the absence of the 1.7-kb DNA band (lane 1) compared with the wild-type gene (lane 3). Lanes 2 and 4 do not display any viral DNA, since they represent left-side ganglia. (C) The successful extraction of genomic DNA from left and right ganglia was confirmed by PCR utilizing primers designed for the murine housekeeping gene G3PDH (385 bp).

View larger version (116K): [in a new window]   Figure 4. Injection of FIV(lacZ) into the right TMJ (5 x 106 infectious particles) resulted in successful transduction of primary sensory neurons with the reporter gene ß-galactosidase in trigeminal ganglia ispilateral to the treated joint. (A) ß-galactosidase expression was detected by X-gal histochemistry in sagittal sections of the right-side ganglion (4X), (B) primarily at its posterior and posterolateral regions (20X). (C) Injection of FIV('lac) did not result in ß-galactosidase expression. (D) The X-gal staining was confirmed with immunocytochemistry, with the use of antibodies raised against bacterial ß-galactosidase following FIV(lacZ) injection compared with (E) FIV('lac) treatment.