Purchase this article with an account.
S. L. Donovan, M. A. Dyer; Genetic Rescue of Immature Rod Phenotype in Rb-Deficient Retinae. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4002. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Where Rb lies in the genetic pathway that underlies rod photoreceptor differentiation is unknown. Rod photoreceptors fail to differentiate in the absence of Rb. Instead the cells remain as immature, undifferentiated cells in the ONL and fail to upregulate genes critical for rod development, such as Nrl, Nr2e3 and Crx. Rb-deficient retinal cells also express high levels of Pax-6, which has been proposed as a putative rod repressor by other studies. We hypothesized that the immature cell phenotype could be rescued by expressing early rod genes or reducing Pax6 expression in Rb-deficient retinae.
To test this hypothesis, candidate rod genes or plasmids encoding Pax-6 siRNAs were electroporated in Rb-deficient retinae utilizing an in vitro approach. To express candidate rod genes, we generated a gene-swap vector, pSD-GS, that contained cre recombinase, a YFP reporter gene and a candidate gene, such as Nrl (pSD-GSNrl). Retinae from P0 RbLox/Lox pups were dissected and electroporated with the gene swap vector, in vitro, and maintained in retinal explant culture for 7-10 days, Figure 1. Retinae were dissociated, sorted and analyzed for changes in expression of downstream photoreceptor target genes and retinal progenitor genes. Gene swap vectors without candidate genes (pSD-GS) and YFP- cells served as controls. To reduce Pax-6 expression, a Pax-6 siRNA was subcloned into the pSilencer vector and electroporated into E14 Rb-/- retinae. Retinae were cultured and processed as described above. A scrambled siRNA and empty pSilencer vector served as controls.
Genetic replacement of Nrl following Rb deletion leads to an increase in the expression of genes found in mature rod photoreceptors, such as, gnat1 and rhodopsin, and a decrease in expression of retinal progenitor genes, such as fgf15 and p107.
Genetic rescue is achieved at a molecular level following acute Rb inactivation and replacement of Nrl at P0. These data suggest that Nrl acts downstream of Rb during rod development.
This PDF is available to Subscribers Only