April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
The Nrl Mutant Retina as an Assay System for Cone-Specific Promoter Activity
Author Affiliations & Notes
  • K. A. Lawrence
    Pathology and Immunology, Washington University, St. Louis, Missouri
  • J. C. Corbo
    Pathology and Immunology, Washington University, St. Louis, Missouri
  • Footnotes
    Commercial Relationships  K.A. Lawrence, None; J.C. Corbo, None.
  • Footnotes
    Support  1R01EY18826-01A1 and T32EY13360-08
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5435. doi:
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      K. A. Lawrence, J. C. Corbo; The Nrl Mutant Retina as an Assay System for Cone-Specific Promoter Activity. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5435.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Despite their critical importance in retinal disease, cone photoreceptors are understudied. Analysis of transgenic mice carrying promoter-reporter fusion constructs is currently the gold standard for assaying photoreceptor cis-regulatory activity in vivo. This approach is laborious and time-intensive, making it impractical for detailed promoter dissection. Therefore, we have developed a more efficient system for assaying cone promoter activity using in vivo electroporation of the ‘all-cone’ Nrl mutant retina. The neural retina leucine zipper transcription factor, Nrl, acts as a photoreceptor-specific cell fate switch: progenitors expressing Nrl differentiate as rods while those that do not express Nrl differentiate as cones. Thus, Nrl-null mice exhibit retinas in which rods have been transfated into cones. We have exploited the ‘all-cone’ retina of the Nrl mutant mouse to identify novel cis-regulatory elements (CREs) and cis-motifs in cone genes as part of a larger study focusing on understanding cone-subtype specific gene regulation.

Methods: : A novel computer algorithm developed by Hsiau et al. (PLoS ONE 2:e643) was used to identify putative CREs around cone genes. Initial studies focused on regions upstream of Opn1sw (blue opsin) and Gnb3 (guanine nucleotide-binding protein beta 3). Promoter-DsRed fusion constructs were electroporated into P0 Nrl mutant retinas in vivo. Retinas were allowed to develop in culture and were harvested on day eight. The expression strength of the cone CREs was quantified by measuring fluorescence intensity in the red channel and normalizing it to that of a co-electroporated GFP control.

Results: : We have demonstrated that the Nrl mutant retina can be used as a rapid assay system for cone promoter activity. We have identified a highly conserved block upstream of Gnb3 responsible for driving strong expression in cones. We are now in the process of carefully dissecting this region as well as those upstream of Opn1sw to pinpoint novel cis-motifs.

Conclusions: : By using the Nrl mutant retina to assay cone promoter activity, we are able to identify novel CREs and cis-motifs controlling cone-specific gene expression.

Keywords: photoreceptors • gene/expression • opsins 

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