Analyses of the guanylate cyclase activating protein-1 gene promoter in the developing retina.
OBJECTIVE
To determine the activity, cell specificity, and developmental expression profiles of fragments of the chicken guanylate cyclase activating protein (GCAP)-1 promoter.


METHODS
The intrinsic activities of five GCAP1 promoter-luciferase constructs were measured in transiently transfected primary chicken embryonic retinal cultures. Lentivirus vectors carrying GCAP1 promoter-nlacZ transgenes were used to examine the cell specificities and temporal expression characteristics of selected promoter fragments in developing retina.


RESULTS
Three of the five GCAP1 promoter fragments exhibited significant activity in vitro. The expression characteristics of the promoter fragments in vivo varied as a function of promoter length. Expression of nlacZ driven by the 0.6- and 1.7-kb GCAP1 promoter fragments was first observed on embryonic day (E)12 and was restricted to the inner nuclear layer (INL). By E16, nlacZ staining was also detected in the outer nuclear layer (ONL). Expression of nlacZ driven by the 4.2-kb GCAP1 promoter fragment was not observed until E16 and was restricted to the ONL.


CONCLUSIONS
The general organization of regulatory cis elements within the GCAP1 promoter is different from other photoreceptor-specific gene promoters. Elements located within 0.3 kb upstream of the transcription start point are capable of producing efficient gene expression; however, additional elements located within 4.0 kb upstream of the transcription start point are necessary to confer on the fragment the cell specificity and developmental expression characteristics of the native GCAP1 promoter. The results of the current study show that the lentiviral vector system is a useful tool for the characterization of the promoters of genes expressed in neural retina.
Coleman
Jason E
JE
Department of Neuroscience, College of Medicine, University of Florida McKnight Brain Institute, 100 Newell Drive, Bldg. 59, Gainesville, FL 32610-0255, USA.
Fuchs
Gabriela E
GE
Semple-Rowland
Susan L
SL
D000818:Animals; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D018929:Cell Culture Techniques; D002642:Chick Embryo; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D005822:Genetic Vectors; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins; D007763:Lac Operon / Q000235:genetics; D016086:Lentivirus / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012160:Retina / Q000196:embryology*; D012168:Retinal Pigments / Q000235:genetics / Q000378:metabolism; D017299:Rod Opsins; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D012168:Retinal Pigments; D017299:Rod Opsins; C034941:iodopsin; D008156:Luciferases; D001616:beta-Galactosidase; D006162:Guanylate Cyclase