PURPOSE. Although the RGC-5 cell line is widely used in retinal ganglion cell (RGC) research, recent data have raised questions about the nature of these cells. The authors performed a systematic analysis of RGC-5 cells to determine which RGC or neuronal markers are expressed after treatment with known differentiating agents, thus providing further insight into the nature of these cells and assisting in defining their future use. METHODS. RGC-5 cells were treated for 5 days with staurosporine (STSN; 316 nM), trichostatin A (TSA; 500 nM), or succinyl-concanavalin A (sConA; 50 microg/mL), after which they were assayed for specific marker antigen/mRNA expression. Treated cells were also assayed for excitotoxic responsiveness. RESULTS. Neither treated nor untreated RGC-5 cells expressed any specific RGC marker mRNAs or proteins (Brn-3, neurofilaments, Thy-1) or calbindin, calretinin, synaptophysin, PKCalpha, or glial fibrillary acidic protein. However, control RGC-5 cells did express the neuronal markers tau, betaIII-tubulin, microtubule-associated protein (MAP)-1b, MAP2, and PGP9.5. Although treatment with sConA had no effect on the expression of these markers, STSN and (dose dependently) TSA increased their expression and induced excitotoxic responsiveness. All cells, treated or not, expressed high levels of nestin but no other progenitor cell markers. All cells also expressed cone-specific, but not rod-specific, opsin indicative of cone photoreceptor lineage. CONCLUSIONS. RGC-5 cells expressed neuronal, but not RGC-specific, markers that were dose dependently upregulated by TSA. Hence, TSA provided the best tested means to terminally differentiate the cells to a neuronal phenotype from a precursor-like lineage.
BackgroundGenetic models have been developed in divergent branches of the class Alphaproteobacteria to help answer a wide spectrum of questions regarding bacterial physiology. For example, Sinorhizobium meliloti serves as a useful representative for investigating rhizobia-plant symbiosis and nitrogen fixation, Caulobacter crescentus for studying cell cycle regulation and organelle biogenesis, and Zymomonas mobilis for assessing the potentials of metabolic engineering and biofuel production. A tightly regulated promoter that enables titratable expression of a cloned gene in these different models is highly desirable, as it can facilitate observation of phenotypes that would otherwise be obfuscated by leaky expression.ResultsWe compared the functionality of four promoter regions in S. meliloti (ParaA, PtauA, PrhaR, and PmelA) by constructing strains carrying fusions to the uidA reporter in their genomes and measuring beta-glucuronidase activities when they were induced by arabinose, taurine, rhamnose, or melibiose. PtauA was chosen for further study because it, and, to a lesser extent, PmelA, exhibited characteristics suitable for efficient modulation of gene expression. The levels of expression from PtauA depended on the concentrations of taurine, in both complex and defined media, in S. meliloti as well as C. crescentus and Z. mobilis. Moreover, our analysis indicated that TauR, TauC, and TauY are each necessary for taurine catabolism and substantiated their designated roles as a transcriptional activator, the permease component of an ABC transporter, and a major subunit of the taurine dehydrogenase, respectively. Finally, we demonstrated that PtauA can be used to deplete essential cellular factors in S. meliloti, such as the PleC histidine kinase and TatB, a component of the twin-arginine transport machinery.ConclusionsThe PtauA promoter of S. meliloti can control gene expression with a relatively inexpensive and permeable inducer, taurine, in diverse alpha-proteobacteria. Regulated expression of the same gene in different hosts can be achieved by placing both tauR and PtauA on appropriate vectors, thus facilitating inspection of conservation of gene function across species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0295-2) contains supplementary material, which is available to authorized users.
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