A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium

Natalie J. Dorà, Martine Manuel, Dirk-Jan Kleinjan, David J. Price, J. Martin Collinson, Robert E. Hill, John D. West (Corresponding Author)

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Abstract

Objective: The corneas of heterozygous Pax6 +/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6 +/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types.
Results: In aA preliminary study, showed that ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos could affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6 fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/- ;RCE:loxP reporter mice was also patchy. We attribute these findingspatchy Pax6 staining to mosaic transgene expression, causing mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6 fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.
Original languageEnglish
Article number705
JournalBMC Research Notes
Volume11
DOIs
Publication statusPublished - 5 Oct 2018

Fingerprint

Corneal Epithelium
Tamoxifen
Stem cells
Staining and Labeling
Transgenes
Stem Cells
Epithelial Cells
Deterioration
Cornea
Embryonic Structures
Alleles
Parturition

Keywords

  • cornea
  • corneal epithelium
  • Cre-loxP
  • CAG-CreER
  • Krt19-CreER
  • Pax6
  • mouse
  • mosaic transgene expression

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A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium. / Dorà, Natalie J.; Manuel, Martine; Kleinjan, Dirk-Jan; Price, David J.; Collinson, J. Martin; Hill, Robert E. ; West, John D. (Corresponding Author).

In: BMC Research Notes, Vol. 11, 705, 05.10.2018.

Research output: Contribution to journalArticle

Dorà, Natalie J. ; Manuel, Martine ; Kleinjan, Dirk-Jan ; Price, David J. ; Collinson, J. Martin ; Hill, Robert E. ; West, John D. / A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium. In: BMC Research Notes. 2018 ; Vol. 11.
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abstract = "Objective: The corneas of heterozygous Pax6 +/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6 +/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types.Results: In aA preliminary study, showed that ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos could affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6 fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/- ;RCE:loxP reporter mice was also patchy. We attribute these findingspatchy Pax6 staining to mosaic transgene expression, causing mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6 fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.",
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T1 - A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium

AU - Dorà, Natalie J.

AU - Manuel, Martine

AU - Kleinjan, Dirk-Jan

AU - Price, David J.

AU - Collinson, J. Martin

AU - Hill, Robert E.

AU - West, John D.

N1 - This work was supported by the Wellcome Trust (grant 088876/Z/09/Z to JDW, REH and JMC). The funding body had no role in the design of the study, the collection, analysis and interpretation of data or the preparation of the manuscript

PY - 2018/10/5

Y1 - 2018/10/5

N2 - Objective: The corneas of heterozygous Pax6 +/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6 +/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types.Results: In aA preliminary study, showed that ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos could affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6 fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/- ;RCE:loxP reporter mice was also patchy. We attribute these findingspatchy Pax6 staining to mosaic transgene expression, causing mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6 fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.

AB - Objective: The corneas of heterozygous Pax6 +/- mice develop abnormally and deteriorate further after birth but it is not known whether the postnatal deterioration is predetermined by abnormal development. Our objective was to identify whether depletion of Pax6 in adult mice caused any corneal abnormalities, similar to those in Pax6 +/- mice, where Pax6 levels are low throughout development and adulthood. We used two tamoxifen-inducible, Cre-loxP experimental strategies to deplete Pax6 either ubiquitously or in a restricted range of cell types.Results: In aA preliminary study, showed that ubiquitous depletion of Pax6 by tamoxifen treatment of E9.5 CAG-CreERTg/-;Pax6fl/fl embryos could affected eye development. Tamoxifen treatment of 12-week old, adult CAG-CreERTg/-;Pax6fl/+ and CAG-CreERTg/-;Pax6 fl/fl mice resulted in weak and/or patchy Pax6 immunostaining in the corneal epithelium but caused no corneal abnormalities. GFP staining in tamoxifen-treated CAG-CreERTg/- ;RCE:loxP reporter mice was also patchy. We attribute these findingspatchy Pax6 staining to mosaic transgene expression, causing mosaic deletion of the Pax6fl allele, probably caused by mosaic CAG-CreERTg expression. In a parallel study, we treated adult Krt19-CreERTg/-;Pax6 fl/+ mice with tamoxifen to try to deplete Pax6 in limbal epithelial stem cells (LESCs) which replenish the corneal epithelium. However, Pax6 staining remained strong after a 12-week chase period so the Krt19-CreERTg/- transgene may have failed to target LESCs.

KW - cornea

KW - corneal epithelium

KW - Cre-loxP

KW - CAG-CreER

KW - Krt19-CreER

KW - Pax6

KW - mouse

KW - mosaic transgene expression

U2 - 10.1186/s13104-018-3812-9

DO - 10.1186/s13104-018-3812-9

M3 - Article

VL - 11

JO - BMC Research Notes

JF - BMC Research Notes

SN - 1756-0500

M1 - 705

ER -