Impairment of rod cGMP-gated channel alpha-subunit expression leads to photoreceptor and bipolar cell degeneration.
OBJECTIVE
To determine whether alterations in rod cGMP-gated channel function mediate retinal degeneration, a transgenic approach in which the alpha subunit of the rod cGMP-gated channel is reduced by the expression of an antisense RNA was used.


METHODS
A 890-bp fragment of the 5' mouse rod cGMP-gated channel cDNA was cloned in the antisense orientation under the control of the strong bacterial cytomegalovirus promoter. This antisense construct was used to generate transgenic mice in which the expression of the antisense transgene was measured by reverse transcription-polymerase chain reaction. Histologic, immunocytochemical, and TdT-dUTP terminal nick-end labeling (TUNEL) analyses were performed on control and transgenic retina sections to determine the effects of antisense expression on specific cell types.


RESULTS
The antisense RNA was able to inhibit the translation of the rod channel protein in an in vitro assay. Three transgenic mouse lines expressing the antisense construct were obtained. Molecular analyses showed that the antisense is expressed in the eye of each transgenic mouse line, where it reduces rod cGMP-gated channel RNA expression. Histologic and immunocytochemical data showed that transgenic retinas have a reduced number of photoreceptors and bipolar cells. TUNEL staining confirmed that photoreceptor and bipolar cells degenerate along an apoptotic pathway.


CONCLUSIONS
Impairment of rod cGMP-gated channel alpha subunit expression leads to photoreceptor and bipolar cell degeneration. These transgenic mice are the first model of retinal degeneration caused by an alteration in the expression of the rod cGMP-gated channel. This model system can be used to test therapies designed to slow or stalled retinal degenerations.
Leconte
L
L
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06520-8061, USA.
Barnstable
C J
CJ
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D015152:Blotting, Northern; D006152:Cyclic GMP / Q000235:genetics* / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D020287:In Situ Nick-End Labeling; D007395:Interneurons / Q000378:metabolism / Q000473:pathology*; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism / Q000473:pathology*; D012313:RNA / Q000378:metabolism*; D016372:RNA, Antisense / Q000378:metabolism; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D054815:Cyclic Nucleotide-Gated Cation Channels; D005136:Eye Proteins; D007473:Ion Channels; D016372:RNA, Antisense; D012313:RNA; D006152:Cyclic GMP