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Spatiotemporal gene targeting in the mouse corneal endothelium

PURPOSE: The inducible Cre-ERT2 recombinase system allows for temporal control of gene targeting, and it is useful to studying adult function of genes that have critical developmental roles. The Zeb1(flox/flox): UBC-CreERT2 mouse was generated to conditionally target Zeb1 to investigate its role in...

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Autores principales: Lee, JeongGoo, Heur, Martin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Wolters Kluwer - Medknow 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10220442/
https://www.ncbi.nlm.nih.gov/pubmed/37252171
http://dx.doi.org/10.4103/tjo.TJO-D-22-00125
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author Lee, JeongGoo
Heur, Martin
author_facet Lee, JeongGoo
Heur, Martin
author_sort Lee, JeongGoo
collection PubMed
description PURPOSE: The inducible Cre-ERT2 recombinase system allows for temporal control of gene targeting, and it is useful to studying adult function of genes that have critical developmental roles. The Zeb1(flox/flox): UBC-CreERT2 mouse was generated to conditionally target Zeb1 to investigate its role in mesenchymal transition in the mouse corneal endothelium in vivo. MATERIALS AND METHODS: Hemizygous UBC-CreERT2 mice were crossed with homozygous mice harboring loxP-flanked Zeb1 alleles (Zeb1(flox/flox)) to generate the Zeb1(flox/flox): UBC-CreERT2 mouse. 4-hydroxytamoxifen (4-OHT) exposure leads to excision of exon 6 of Zeb1, resulting in a loss function allele in the Zeb1(flox/flox): UBC-CreERT2 mouse. Intracameral 4-OHT injection further isolates Zeb1 targeting to the anterior chamber. Mesenchymal transition and induction of Zeb1 expression in the corneal endothelium was achieved using FGF2 in ex vivo organ culture. Gene expression was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and by immunoblotting in the mouse corneal endothelium in vivo. RESULTS: Following Cre-mediated targeting of Zeb1 by intracameral 4-OHT injection in Zeb1(flox/flox): UBC-CreERT2 mice, FGF2 treatment in ex vivo organ culture resulted in abrogation of Zeb1 mRNA and protein expression in the corneal endothelium. CONCLUSION: The data show Zeb1, a critical mediator of fibrosis in corneal endothelial mesenchymal transition, can be targeted by intracameral injection of 4-OHT in the mouse corneal endothelium in vivo. These results suggest that genes with critical developmental roles can be targeted at a specific time in the corneal endothelium to study its role in adult disease using an inducible Cre-Lox strategy.
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spelling pubmed-102204422023-05-28 Spatiotemporal gene targeting in the mouse corneal endothelium Lee, JeongGoo Heur, Martin Taiwan J Ophthalmol Original Article PURPOSE: The inducible Cre-ERT2 recombinase system allows for temporal control of gene targeting, and it is useful to studying adult function of genes that have critical developmental roles. The Zeb1(flox/flox): UBC-CreERT2 mouse was generated to conditionally target Zeb1 to investigate its role in mesenchymal transition in the mouse corneal endothelium in vivo. MATERIALS AND METHODS: Hemizygous UBC-CreERT2 mice were crossed with homozygous mice harboring loxP-flanked Zeb1 alleles (Zeb1(flox/flox)) to generate the Zeb1(flox/flox): UBC-CreERT2 mouse. 4-hydroxytamoxifen (4-OHT) exposure leads to excision of exon 6 of Zeb1, resulting in a loss function allele in the Zeb1(flox/flox): UBC-CreERT2 mouse. Intracameral 4-OHT injection further isolates Zeb1 targeting to the anterior chamber. Mesenchymal transition and induction of Zeb1 expression in the corneal endothelium was achieved using FGF2 in ex vivo organ culture. Gene expression was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and by immunoblotting in the mouse corneal endothelium in vivo. RESULTS: Following Cre-mediated targeting of Zeb1 by intracameral 4-OHT injection in Zeb1(flox/flox): UBC-CreERT2 mice, FGF2 treatment in ex vivo organ culture resulted in abrogation of Zeb1 mRNA and protein expression in the corneal endothelium. CONCLUSION: The data show Zeb1, a critical mediator of fibrosis in corneal endothelial mesenchymal transition, can be targeted by intracameral injection of 4-OHT in the mouse corneal endothelium in vivo. These results suggest that genes with critical developmental roles can be targeted at a specific time in the corneal endothelium to study its role in adult disease using an inducible Cre-Lox strategy. Wolters Kluwer - Medknow 2023-01-11 /pmc/articles/PMC10220442/ /pubmed/37252171 http://dx.doi.org/10.4103/tjo.TJO-D-22-00125 Text en Copyright: © 2023 Taiwan Journal of Ophthalmology https://creativecommons.org/licenses/by-nc-sa/4.0/This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
spellingShingle Original Article
Lee, JeongGoo
Heur, Martin
Spatiotemporal gene targeting in the mouse corneal endothelium
title Spatiotemporal gene targeting in the mouse corneal endothelium
title_full Spatiotemporal gene targeting in the mouse corneal endothelium
title_fullStr Spatiotemporal gene targeting in the mouse corneal endothelium
title_full_unstemmed Spatiotemporal gene targeting in the mouse corneal endothelium
title_short Spatiotemporal gene targeting in the mouse corneal endothelium
title_sort spatiotemporal gene targeting in the mouse corneal endothelium
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10220442/
https://www.ncbi.nlm.nih.gov/pubmed/37252171
http://dx.doi.org/10.4103/tjo.TJO-D-22-00125
work_keys_str_mv AT leejeonggoo spatiotemporalgenetargetinginthemousecornealendothelium
AT heurmartin spatiotemporalgenetargetinginthemousecornealendothelium