A system for inducible gene expression in retinal ganglion cells

John B. Kerrison, Elia J Duh, Yilin Yu, Deborah C. Otteson, Donald J Zack

Research output: Contribution to journalArticle

Abstract

PURPOSE. To develop a system for inducible gene expression in retinal ganglion cells, Thy1 and ckit promoters were used to direct expression of a second-generation reverse tetracycline transactivator (rtTA2(S)-M2). METHODS. Transgenic mice were generated that harbor rtTA2(S)-M2 under the control of either the Thy1 or ckit promoter. These animals were crossed with mice transgenic for the LacZ gene downstream of a cassette of tet operator (TRE) binding sites. Induction of the LacZ reporter gene in vivo after either oral or subcutaneous doxycycline administration and in vitro in cultured retinal cells was assessed. To examine induction of a secreted protein, expression of pigment epithelium-derived factor (PEDF) in mice harboring Thy1-rtTA and TRE-PEDF constructs was quantified. RESULTS. Five of seven Thy1-rtTA lines showed induction with subcutaneous doxycycline: maximum induction in one line (Thy1-C), moderate in one line (Thy1-F), and minimal in three lines. There was no detectable retinal LacZ expression in the ckit-rtTA lines, despite expression of the ckit-rtTA transgene at the RNA level. In Thy1-rtTA lines, LacZ reporter expression as measured by X-gal staining was evenly dispersed throughout all quadrants of the retina, present in a subpopulation of retinal ganglion cell (RGC) bodies, RGC axons projecting through the retina and optic nerve, and some cells in the inner nuclear layer. Immunostaining for β-galactosidase demonstrated more uniform expression in RGCs and cells of the inner aspect of the inner nuclear layer, which, by double staining with anti-β- galactosidase and anti-calretinin antibodies, were consistent with amacrine cells. More than 95% of Thy-1 antigen-positive cells in the retina expressed the induced transgene. Subcutaneous doxycycline resulted in a more robust induction of LacZ than did oral administration. In vitro, the number of cells induced in culture increased in a dose-dependent manner, with maximum expression at 10 μg/mL at a level 3.4-fold over background. Thy1-rtTA/TRE-PEDF mice treated with doxycycline had 1000-fold induction in their retinal PEDF expression in comparison with nontransgenic mice and 600-fold induction over noninduced Thy1-rtTA/TRE-PEDF mice. CONCLUSIONS. A transgenic system for inducible RGC expression has been developed that demonstrates minimal leakiness and significant induction with doxycycline. This system will be useful for several applications.

Original languageEnglish (US)
Pages (from-to)2932-2939
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume46
Issue number8
DOIs
StatePublished - 2005

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Retinal Ganglion Cells
Doxycycline
Gene Expression
Galactosidases
Retina
Lac Operon
Transgenes
Transgenic Mice
Thy-1 Antigens
Staining and Labeling
Calbindin 2
Amacrine Cells
Trans-Activators
Retinal Pigment Epithelium
Optic Nerve
Tetracycline
Reporter Genes
Oral Administration
Axons
Anti-Idiotypic Antibodies

ASJC Scopus subject areas

  • Ophthalmology

Cite this

A system for inducible gene expression in retinal ganglion cells. / Kerrison, John B.; Duh, Elia J; Yu, Yilin; Otteson, Deborah C.; Zack, Donald J.

In: Investigative Ophthalmology and Visual Science, Vol. 46, No. 8, 2005, p. 2932-2939.

Research output: Contribution to journalArticle

Kerrison, John B. ; Duh, Elia J ; Yu, Yilin ; Otteson, Deborah C. ; Zack, Donald J. / A system for inducible gene expression in retinal ganglion cells. In: Investigative Ophthalmology and Visual Science. 2005 ; Vol. 46, No. 8. pp. 2932-2939.
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AU - Duh, Elia J

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AU - Otteson, Deborah C.

AU - Zack, Donald J

PY - 2005

Y1 - 2005

N2 - PURPOSE. To develop a system for inducible gene expression in retinal ganglion cells, Thy1 and ckit promoters were used to direct expression of a second-generation reverse tetracycline transactivator (rtTA2(S)-M2). METHODS. Transgenic mice were generated that harbor rtTA2(S)-M2 under the control of either the Thy1 or ckit promoter. These animals were crossed with mice transgenic for the LacZ gene downstream of a cassette of tet operator (TRE) binding sites. Induction of the LacZ reporter gene in vivo after either oral or subcutaneous doxycycline administration and in vitro in cultured retinal cells was assessed. To examine induction of a secreted protein, expression of pigment epithelium-derived factor (PEDF) in mice harboring Thy1-rtTA and TRE-PEDF constructs was quantified. RESULTS. Five of seven Thy1-rtTA lines showed induction with subcutaneous doxycycline: maximum induction in one line (Thy1-C), moderate in one line (Thy1-F), and minimal in three lines. There was no detectable retinal LacZ expression in the ckit-rtTA lines, despite expression of the ckit-rtTA transgene at the RNA level. In Thy1-rtTA lines, LacZ reporter expression as measured by X-gal staining was evenly dispersed throughout all quadrants of the retina, present in a subpopulation of retinal ganglion cell (RGC) bodies, RGC axons projecting through the retina and optic nerve, and some cells in the inner nuclear layer. Immunostaining for β-galactosidase demonstrated more uniform expression in RGCs and cells of the inner aspect of the inner nuclear layer, which, by double staining with anti-β- galactosidase and anti-calretinin antibodies, were consistent with amacrine cells. More than 95% of Thy-1 antigen-positive cells in the retina expressed the induced transgene. Subcutaneous doxycycline resulted in a more robust induction of LacZ than did oral administration. In vitro, the number of cells induced in culture increased in a dose-dependent manner, with maximum expression at 10 μg/mL at a level 3.4-fold over background. Thy1-rtTA/TRE-PEDF mice treated with doxycycline had 1000-fold induction in their retinal PEDF expression in comparison with nontransgenic mice and 600-fold induction over noninduced Thy1-rtTA/TRE-PEDF mice. CONCLUSIONS. A transgenic system for inducible RGC expression has been developed that demonstrates minimal leakiness and significant induction with doxycycline. This system will be useful for several applications.

AB - PURPOSE. To develop a system for inducible gene expression in retinal ganglion cells, Thy1 and ckit promoters were used to direct expression of a second-generation reverse tetracycline transactivator (rtTA2(S)-M2). METHODS. Transgenic mice were generated that harbor rtTA2(S)-M2 under the control of either the Thy1 or ckit promoter. These animals were crossed with mice transgenic for the LacZ gene downstream of a cassette of tet operator (TRE) binding sites. Induction of the LacZ reporter gene in vivo after either oral or subcutaneous doxycycline administration and in vitro in cultured retinal cells was assessed. To examine induction of a secreted protein, expression of pigment epithelium-derived factor (PEDF) in mice harboring Thy1-rtTA and TRE-PEDF constructs was quantified. RESULTS. Five of seven Thy1-rtTA lines showed induction with subcutaneous doxycycline: maximum induction in one line (Thy1-C), moderate in one line (Thy1-F), and minimal in three lines. There was no detectable retinal LacZ expression in the ckit-rtTA lines, despite expression of the ckit-rtTA transgene at the RNA level. In Thy1-rtTA lines, LacZ reporter expression as measured by X-gal staining was evenly dispersed throughout all quadrants of the retina, present in a subpopulation of retinal ganglion cell (RGC) bodies, RGC axons projecting through the retina and optic nerve, and some cells in the inner nuclear layer. Immunostaining for β-galactosidase demonstrated more uniform expression in RGCs and cells of the inner aspect of the inner nuclear layer, which, by double staining with anti-β- galactosidase and anti-calretinin antibodies, were consistent with amacrine cells. More than 95% of Thy-1 antigen-positive cells in the retina expressed the induced transgene. Subcutaneous doxycycline resulted in a more robust induction of LacZ than did oral administration. In vitro, the number of cells induced in culture increased in a dose-dependent manner, with maximum expression at 10 μg/mL at a level 3.4-fold over background. Thy1-rtTA/TRE-PEDF mice treated with doxycycline had 1000-fold induction in their retinal PEDF expression in comparison with nontransgenic mice and 600-fold induction over noninduced Thy1-rtTA/TRE-PEDF mice. CONCLUSIONS. A transgenic system for inducible RGC expression has been developed that demonstrates minimal leakiness and significant induction with doxycycline. This system will be useful for several applications.

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