Conditional regulation of Hoxa2 gene expression in CG4 cells
Wang, Juan (Monica)
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.
DegreeMaster of Science (M.Sc.)
SupervisorNazarali, Adil J.
CG4 cell line