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Concepcion Lillo, Saúl Herranz-Martín, Antonio E. Paniagua, Almudena Velasco, Juan M. Lara, José Aijón; Phenotype of the Müller Cells of the Crb1rd8 Mutant Mouse in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2007.
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© ARVO (1962-2015); The Authors (2016-present)
Müller cells are the radial glial of the retina, and at the level of the retinal outer limiting membrane (OLM), they establish adherens junctions with photoreceptor cells. CRB1 is a transmembrane protein present in the Müller cells, and localized in the subapical region (SAR) of the OLM. Mutations in the gene that codifies for the CRB1 human protein lead to retinal degenerations, such as Leber Congenial Amaurosis or Retinitis pigmentosa. The Crb1rd8 mouse has a mutation identified as a deletion of a single base in the Crb1 gene, encoding a truncated protein. There is a clear phenotype in the Crb1rd8 mouse retina, showing gaps in the OLM, photoreceptor-rosettes and progressive photoreceptor death. Nevertheless, little is known about the characteristics of the Müller cells in the mutant retina. To characterize the modifications that the CRB1 mutant Müller cells may experiment during the development of the retina, our purpose is to analyze their behavior in vitro as well as in vivo comparing wild type and mutant cells.
Müller cells from mutant and wild type mouse retinas were isolated at P7, cultured, and their viability was examined at different time points. We performed western blot and immunofluorescence analysis with well-known markers for this cell type in the cells in vitro and in the retinal tissue. Finally, we have tested the possibility of transfecting Müller cells with Chariot©, a commercial agent that introduce proteins in their native configuration in cells in vitro and in vivo.
Müller cells primary cell cultures are viable at seven days in culture. We found differences in the viability of the mutant cells compared with the wild type cells at the different time points measured. Besides, there are also variations in the amount of expression of several Müller cells markers, such as GFAP, glutamine synthetase and SOX2 comparing wild type and mutant Müller cells in vivo and in vitro. We have detected these differences from early development of the retina, being more evident in the primary cultures. We have used the commercial transfection agent Chariot© to introduce b-galactosidase in Müller cells in vitro and in vivo from both phenotypes.
We have characterized the phenotype of the CRB1 mutant Müller cells in vivo and in vitro from the development of the retina onwards. These cells are viable in vitro and, as well as in vivo, they show a pattern of expression of several Müller cells markers which is different to that of the wild type ones. Finally, we have verified that Müller cells obtained from both phenotypes, mutant and control, are susceptible of being transfected with the commercial agent Chariot© with a high efficiency.
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