Oligodendrocytes (OLs) are the glial cells responsible for the synthesis and maintenance of myelin in the central nervous system. Recently, Hoxa2 was found by our laboratory to be expressed by OLs and down-regulated at the terminal differentiation stage during oligodendrogenesis in mice (Nicolay et al., 2004b). To further investigate the role of Hoxa2 in oligodendroglial development, a tetracycline regulated controllable expression system was utilized to establish two stable cell lines where the expression level of Hoxa2 gene could be up-regulated (CG4-SHoxa2 [sense Hoxa2]) or down-regulated (CG4-ASHoxa2 [Antisense Hoxa2]) in CG4 glial cells. Morphologically, no obvious differences were observed between CG4-SHoxa2 and CG4 wild-type cells, whereas CG4-ASHoxa2 cells exhibited much shorter processes compared with those of CG4 wild-type cells. Data from BrdU uptake assays indicated that an up-regulation of Hoxa2 gene promoted the proliferation of CG4-SHoxa2 cells. PDGF&alphaR (Platelet-derived growth factor [PDGF] receptor alpha), a receptor for the mitogen PDGF that enhances the survival and proliferation of OLs, was assessed at the mRNA level in both CG4 and CG4-SHoxa2 cells, but no significant differences were observed between Hoxa2 up-regulated cells and wild-type CG4 cells with respect to the mRNA level of PDGF&alphaR. In addition, specific investigations of the differentiation of CG4-SHoxa2 cells were carried out by characterizing the composition of stage specific oligodendroglial subpopulations in culture. Our immunocytochemical study did not indicate the differentiation course of the genetically engineered cells was significantly altered compared to CG4 wild-type cells, although results from semi-quantitative RT-PCR of oligodendrocyte-specific ceramide galactosyltransferase (CGT) and myelin basic protein (MBP) indicate that the differentiation of CG4-SHoxa2 cells was delayed when Hoxa2 gene was up-regulated.

Oligodendrocytes (OLs) are the glial cells responsible for the synthesis and maintenance of myelin in the central nervous system. Recently, Hoxa2 was found by our laboratory to be expressed by OLs and down-regulated at the terminal differentiation stage during oligodendrogenesis in mice (Nicolay et al., 2004b). To further investigate the role of Hoxa2 in oligodendroglial development, a tetracycline regulated controllable expression system was utilized to establish two stable cell lines where the expression level of Hoxa2 gene could be up-regulated (CG4-SHoxa2 [sense Hoxa2]) or down-regulated (CG4-ASHoxa2 [Antisense Hoxa2]) in CG4 glial cells. Morphologically, no obvious differences were observed between CG4-SHoxa2 and CG4 wild-type cells, whereas CG4-ASHoxa2 cells exhibited much shorter processes compared with those of CG4 wild-type cells. Data from BrdU uptake assays indicated that an up-regulation of Hoxa2 gene promoted the proliferation of CG4-SHoxa2 cells. PDGF&alphaR (Platelet-derived growth factor [PDGF] receptor alpha), a receptor for the mitogen PDGF that enhances the survival and proliferation of OLs, was assessed at the mRNA level in both CG4 and CG4-SHoxa2 cells, but no significant differences were observed between Hoxa2 up-regulated cells and wild-type CG4 cells with respect to the mRNA level of PDGF&alphaR. In addition, specific investigations of the differentiation of CG4-SHoxa2 cells were carried out by characterizing the composition of stage specific oligodendroglial subpopulations in culture. Our immunocytochemical study did not indicate the differentiation course of the genetically engineered cells was significantly altered compared to CG4 wild-type cells, although results from semi-quantitative RT-PCR of oligodendrocyte-specific ceramide galactosyltransferase (CGT) and myelin basic protein (MBP) indicate that the differentiation of CG4-SHoxa2 cells was delayed when Hoxa2 gene was up-regulated.

Two common strategies used to identify specific genes that influence aging in Drosophila melanogaster are overexpression screens and candidate gene approaches. Both of these strategies rely on gene expression systems. A very popular gene expression system in Drosophila is the bipartite UAS/GAL4 system, where binding of GAL4 to a UAS sequence can direct the expression of a UAS-linked transgene in a pattern determined by GAL4. Although the UAS/GAL4 system allows for spatial regulation of transgene expression, it does not allow researchers to control when transgene expression will occur. This is an important consideration since aging research is primarily interested in identifying genes that influence aging during adulthood, therefore requiring that transgene expression be effectively blocked during pre-adult stages. Both the Gene-Switch and the Tet-Off/GAL80 systems are attempts to establish temporal control over GAL4 activity. The Gene-Switch system is based on a modified form of GAL4 whose transcriptional activity can be controlled through the antiprogestin molecule RU486. The Tet-Off/GAL80 system, where expression of GAL80 (a negative regulator of GAL4) is under the control of a tetracycline sensitive expression system, allows regulation of GAL4 activity through the antibiotic tetracycline. Characterization of these systems reveals that although neither system can completely repress leaky transgene expression, the Tet-Off/GAL80 system is much better at preventing unwanted transgene expression at most stages of the fly life cycle. Furthermore, comparison of muscle specific GAL4 and Gene-Switch strains revealed that upon treatment with their respective inducers, the Tet-Off/GAL80 system allows for GAL4 activity in the muscles, while the Gene-Switch system results in GAL4 activity in other tissues in addition to the muscles. In other characterized Gene-Switch strains, GAL4 activity is achieved only in a subset of the cells of the targeted tissue, suggesting that the Gene-Switch system may be ill-suited for aging studies. These findings, along with the fact that the Tet-Off/GAL80 but not the Gene-Switch system is compatible with the hundreds of characterized GAL4 lines presently available which allows transgene expression to be targeted to most tissues, indicate that the Tet-Off/GAL80 system is the best-suited for aging studies in Drosophila at present. / Thesis (Ph.D, Biology) -- Queen's University, 2008-12-22 17:13:42.089