Insertion of a Pax6 consensus binding site into the αA-crystallin promoter acts as a lens epithelial cell enhancer in transgenic mice

Haotian Zhao, Ying Yang, Christian M. Rizo, Paul A. Overbeek, Michael L. Robinson

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

PURPOSE. Although the murine αA-crystallin promoter is the most commonly used promoter for achieving transgene expression in the developing lens, this promoter directs transgene expression efficiently only in lens fiber cells. The purpose of the present study was to generate promoters capable of directing transgene expression to the entire lens but not to the corneal epithelium. METHODS: Transgenic mice were generated with fragments of the murine αA- and αB-crystallin promoters, as well as with an αA-crystallin promoter engineered with the insertion of a Pax6 consensus binding site driving either human growth hormone (hGH) or Cre recombinase genes, hGH expression was evaluated by in situ hybridization and immunohistochemistry. Cre expression was revealed by x-gal staining after crossing Cre transgenic mice with a Cre reporter strain. RESULTS. Within the lens, the -214/+38 αB-crystallin promoter fragment directed transgene expression in the lens epithelium, but not in fiber cells. The native -282/+43 αA-crystallin promoter drove transgene expression in the lens fiber cells of several independent lines of transgenic mice, but none of these mice demonstrated significant transgene expression in the lens epithelium. In contrast, the insertion of a 32-bp sequence containing a Pax6 consensus binding site into the -282/+43 αA-crystallin promoter reproducibly led to transgene expression in the lens epithelium as well as the lens fiber cells. CONCLUSIONS. The inclusion of a Pax6 consensus binding site within the -282/+43 αA-crystallin promoter enhances the ability of this promoter to drive transgene expression in the lens epithelium.

Original languageEnglish (US)
Pages (from-to)1930-1939
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume45
Issue number6
DOIs
StatePublished - Jun 1 2004

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Crystallins
Transgenic Mice
Lenses
Transgenes
Epithelial Cells
Binding Sites
Epithelium
Human Growth Hormone
Corneal Epithelium
In Situ Hybridization
Immunohistochemistry
Staining and Labeling

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Insertion of a Pax6 consensus binding site into the αA-crystallin promoter acts as a lens epithelial cell enhancer in transgenic mice. / Zhao, Haotian; Yang, Ying; Rizo, Christian M.; Overbeek, Paul A.; Robinson, Michael L.

In: Investigative Ophthalmology and Visual Science, Vol. 45, No. 6, 01.06.2004, p. 1930-1939.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. Although the murine αA-crystallin promoter is the most commonly used promoter for achieving transgene expression in the developing lens, this promoter directs transgene expression efficiently only in lens fiber cells. The purpose of the present study was to generate promoters capable of directing transgene expression to the entire lens but not to the corneal epithelium. METHODS: Transgenic mice were generated with fragments of the murine αA- and αB-crystallin promoters, as well as with an αA-crystallin promoter engineered with the insertion of a Pax6 consensus binding site driving either human growth hormone (hGH) or Cre recombinase genes, hGH expression was evaluated by in situ hybridization and immunohistochemistry. Cre expression was revealed by x-gal staining after crossing Cre transgenic mice with a Cre reporter strain. RESULTS. Within the lens, the -214/+38 αB-crystallin promoter fragment directed transgene expression in the lens epithelium, but not in fiber cells. The native -282/+43 αA-crystallin promoter drove transgene expression in the lens fiber cells of several independent lines of transgenic mice, but none of these mice demonstrated significant transgene expression in the lens epithelium. In contrast, the insertion of a 32-bp sequence containing a Pax6 consensus binding site into the -282/+43 αA-crystallin promoter reproducibly led to transgene expression in the lens epithelium as well as the lens fiber cells. CONCLUSIONS. The inclusion of a Pax6 consensus binding site within the -282/+43 αA-crystallin promoter enhances the ability of this promoter to drive transgene expression in the lens epithelium.",
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N2 - PURPOSE. Although the murine αA-crystallin promoter is the most commonly used promoter for achieving transgene expression in the developing lens, this promoter directs transgene expression efficiently only in lens fiber cells. The purpose of the present study was to generate promoters capable of directing transgene expression to the entire lens but not to the corneal epithelium. METHODS: Transgenic mice were generated with fragments of the murine αA- and αB-crystallin promoters, as well as with an αA-crystallin promoter engineered with the insertion of a Pax6 consensus binding site driving either human growth hormone (hGH) or Cre recombinase genes, hGH expression was evaluated by in situ hybridization and immunohistochemistry. Cre expression was revealed by x-gal staining after crossing Cre transgenic mice with a Cre reporter strain. RESULTS. Within the lens, the -214/+38 αB-crystallin promoter fragment directed transgene expression in the lens epithelium, but not in fiber cells. The native -282/+43 αA-crystallin promoter drove transgene expression in the lens fiber cells of several independent lines of transgenic mice, but none of these mice demonstrated significant transgene expression in the lens epithelium. In contrast, the insertion of a 32-bp sequence containing a Pax6 consensus binding site into the -282/+43 αA-crystallin promoter reproducibly led to transgene expression in the lens epithelium as well as the lens fiber cells. CONCLUSIONS. The inclusion of a Pax6 consensus binding site within the -282/+43 αA-crystallin promoter enhances the ability of this promoter to drive transgene expression in the lens epithelium.

AB - PURPOSE. Although the murine αA-crystallin promoter is the most commonly used promoter for achieving transgene expression in the developing lens, this promoter directs transgene expression efficiently only in lens fiber cells. The purpose of the present study was to generate promoters capable of directing transgene expression to the entire lens but not to the corneal epithelium. METHODS: Transgenic mice were generated with fragments of the murine αA- and αB-crystallin promoters, as well as with an αA-crystallin promoter engineered with the insertion of a Pax6 consensus binding site driving either human growth hormone (hGH) or Cre recombinase genes, hGH expression was evaluated by in situ hybridization and immunohistochemistry. Cre expression was revealed by x-gal staining after crossing Cre transgenic mice with a Cre reporter strain. RESULTS. Within the lens, the -214/+38 αB-crystallin promoter fragment directed transgene expression in the lens epithelium, but not in fiber cells. The native -282/+43 αA-crystallin promoter drove transgene expression in the lens fiber cells of several independent lines of transgenic mice, but none of these mice demonstrated significant transgene expression in the lens epithelium. In contrast, the insertion of a 32-bp sequence containing a Pax6 consensus binding site into the -282/+43 αA-crystallin promoter reproducibly led to transgene expression in the lens epithelium as well as the lens fiber cells. CONCLUSIONS. The inclusion of a Pax6 consensus binding site within the -282/+43 αA-crystallin promoter enhances the ability of this promoter to drive transgene expression in the lens epithelium.

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