Coupling ex vivo electroporation of mouse retinas and luciferase reporter assays to assess rod-specific promoter activity.
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.
Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, 1004 Lausanne, Switzerland; Institute for Research in Ophthalmology, 1950 Sion, Switzerland.
Arnaud
A
Boulling
Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, 1004 Lausanne, Switzerland. Electronic address: pascal.escher@unil.ch.
Pascal
P
Escher
D000818:Animals; D018274:Electroporation / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D057093:Orphan Nuclear Receptors; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics* / Q000378:metabolism
C485736:Nr2e3 protein, mouse; D057093:Orphan Nuclear Receptors; D012333:RNA, Messenger; D012243:Rhodopsin; D008156:Luciferases
Photoreceptor; Promoter studies; Retinal gene expression; Rod promoter; Transfection