Impairment of rod cGMP-gated channel α-subunit expression leads to photoreceptor and bipolar cell degeneration

Laurence Leconte, Colin Barnstable

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

PURPOSE. To determine whether alterations in rod cGMP-gated channel function mediate retinal degeneration, a transgenic approach in which the α 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 α 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.

Original languageEnglish (US)
Pages (from-to)917-926
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume41
Issue number3
StatePublished - Mar 30 2000

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Photoreceptor Cells
Retinal Degeneration
Transgenic Mice
Antisense RNA
Retina
Cytomegalovirus
Transgenes
Reverse Transcription
Complementary DNA
RNA
Staining and Labeling
Polymerase Chain Reaction
Proteins
Therapeutics

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

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title = "Impairment of rod cGMP-gated channel α-subunit expression leads to photoreceptor and bipolar cell degeneration",
abstract = "PURPOSE. To determine whether alterations in rod cGMP-gated channel function mediate retinal degeneration, a transgenic approach in which the α 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 α 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.",
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Impairment of rod cGMP-gated channel α-subunit expression leads to photoreceptor and bipolar cell degeneration. / Leconte, Laurence; Barnstable, Colin.

In: Investigative Ophthalmology and Visual Science, Vol. 41, No. 3, 30.03.2000, p. 917-926.

Research output: Contribution to journalArticle

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AU - Leconte, Laurence

AU - Barnstable, Colin

PY - 2000/3/30

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N2 - PURPOSE. To determine whether alterations in rod cGMP-gated channel function mediate retinal degeneration, a transgenic approach in which the α 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 α 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.

AB - PURPOSE. To determine whether alterations in rod cGMP-gated channel function mediate retinal degeneration, a transgenic approach in which the α 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 α 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.

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