Coupling ex vivo electroporation of mouse retinas and luciferase reporter assays to assess rod-specific promoter activity

Exp Eye Res. 2016 Jul:148:79-82. doi: 10.1016/j.exer.2016.06.002. Epub 2016 Jun 3.

Abstract

Ex vivo electroporation of mouse retinas is an established tool to modulate gene expression and to study cell type-specific gene expression. Here we coupled ex vivo electroporation to luciferase reporter assays to facilitate the study of rod-photoreceptor-specific gene promoters. The activity of the rod-specific proximal bovine rhodopsin promoter was significantly increased in C57BL/6J wild-type retinas at postnatal days 1 and 7 by 3.4-fold and 8.7-fold respectively. In C57BL/6J Nr2e3(rd7/rd7) retinas, where the rod photoreceptor-specific nuclear receptor Nr2e3 is not expressed, a significant increase by 2.5-fold was only observed at postnatal day 7. Cone-specific S-opsin promoter activity was not modulated in C57BL/6J wild-type and Nr2e3(rd7/rd7) retinas. Taken together, we describe an easily implementable protocol to assess rod-specific promoter activity in a physiological context resembling that of the developing postnatal mouse retina.

Keywords: Photoreceptor; Promoter studies; Retinal gene expression; Rod promoter; Transfection.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Electroporation / methods*
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation*
  • Luciferases / genetics
  • Mice
  • Mice, Inbred C57BL
  • Orphan Nuclear Receptors
  • Promoter Regions, Genetic*
  • RNA, Messenger / metabolism
  • Retina / metabolism*
  • Retinal Rod Photoreceptor Cells / metabolism
  • Rhodopsin / genetics*
  • Rhodopsin / metabolism

Substances

  • Nr2e3 protein, mouse
  • Orphan Nuclear Receptors
  • RNA, Messenger
  • Rhodopsin
  • Luciferases