May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Cone–Specific Expression Driven by X–Arrestin Promoters in Transgenic Models
Author Affiliations & Notes
  • J.P. Marion
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL
  • Z.–Y. Huang
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL
  • T. Fujimaki
    Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • H. Kitagawa
    Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • H. Sakuma
    Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • A. Murakami
    Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • A. Kanai
    Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan
  • G. Inana
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL
  • Footnotes
    Commercial Relationships  J.P. Marion, None; Z. Huang, None; T. Fujimaki, None; H. Kitagawa, None; H. Sakuma, None; A. Murakami, None; A. Kanai, None; G. Inana, None.
  • Footnotes
    Support  Foundation Fighting Blindness, Research to Prevent Blindness, NIH P30 EY014801
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 3069. doi:
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      J.P. Marion, Z.–Y. Huang, T. Fujimaki, H. Kitagawa, H. Sakuma, A. Murakami, A. Kanai, G. Inana; Cone–Specific Expression Driven by X–Arrestin Promoters in Transgenic Models . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3069.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: X–arrestin is an arrestin present specifically in the outer segments of red–, green–, and blue– cone photoreceptors. The human X–arrestin gene is on Xcen–q22, and consists of 17 exons with a promoter containing a TATA box and elements important for photoreceptor expression, including three CRX and one PCE–1 elements. In previous studies, using truncation and site–directed mutagenesis, we tested for promoter activity in retinoblastoma cells by transient expression. We demonstrated that the upstream 378bp region of the gene, containing the CRX and PCE–1 elements, is important for expression in retinoblastoma cells in vitro (Fujimaki et al. Gene 339: 139–147, 2004). In the present study for in vivo analysis of the human X–arrestin promoter, we have generated three lines of transgenic mice with constructs, in which the gene encoding beta–galactosidase (LacZ) is driven by the human 1.0 kbp and 2.0 kbp promoter fragments, and the mouse 1.0 kbp promoter fragment, in order to elucidate the mechanism responsible for the in–vivo pan–cone–specific expression of X–arrestin. Methods: Transgenic mice were produced by injecting constructs carrying the human 1.0 kbp, 2.0 kbp, and the mouse 1.0 kbp promoter fragments of the X–arrestin gene fused to the beta–galactosidase gene. After screening the transgenic mice by PCR and Southern blotting analysis, the identity of the beta–galactosidase–expressing cells was established by immunostaining of the retina with antibodies against rat X–arrestin and beta–galactosidase. Results: Immunofluorescence analysis showed that the human 1.0 kbp promoter fragment did not show beta–galactosidase expression in the transgenic retina. However, the transgenic lines containing the mouse 1.0 kbp and the human 2.0 kbp promoter fragment showed expression of beta–galactosidase activity, localized to the cone outer segments to cone synapses. Conclusions: The result indicated that the 2.0 kbp human X–arrestin promoter fragment, but not the 1.0 kbp fragment, and the mouse 1.0 kbp X–arrestin fragment can direct reporter gene expression specifically in the mouse cone photoreceptors. Thus, the expression patterns of the promoter fragments in vivo are different from that in vitro, attesting to the difference in the control of the X–arrestin promoter in the physiological environment of the mouse cone photoreceptors versus the retinoblastoma cell line in culture.

Keywords: photoreceptors • transgenics/knock-outs • gene/expression 
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