May 2004
Volume 45, Issue 13
ARVO Annual Meeting Abstract  |   May 2004
Thioltransferase Knockout mouse: a new cataract model
Author Affiliations & Notes
  • M.F. Lou
    Veterinary & Biomed Sciences, University of Nebraska–Lincoln, Lincoln, NE
    Department of Ophthalmology, University of Nebraska Medical Center, Omaha, NE
  • K. Xing
    Veterinary & Biomed Sciences, University of Nebraska–Lincoln, Lincoln, NE
  • M.R. Fernando
    Veterinary & Biomed Sciences, University of Nebraska–Lincoln, Lincoln, NE
  • S. Moon
    Veterinary & Biomed Sciences, University of Nebraska–Lincoln, Lincoln, NE
  • Y.–S. Ho
    Institute of Environmental Health Sciences, Wayne State University, Detroit, MI
  • Footnotes
    Commercial Relationships  M.F. Lou, None; K. Xing, None; M.R. Fernando, None; S. Moon, None; Y. Ho, None.
  • Footnotes
    Support  NIH Grant EY10590
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 1026. doi:
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      M.F. Lou, K. Xing, M.R. Fernando, S. Moon, Y.–S. Ho; Thioltransferase Knockout mouse: a new cataract model . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1026.

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

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Abstract: : Purpose: To establish a thioltransferase (TTase, or Grx1) gene knockout (KO) mouse model and use the model to confirm the important role of TTase, the dethiolating enzyme, in defending oxidative stress by regulating protein–S–thiolation and preventing cataract formation. Methods: Genomic clone containing the mouse Grx1 gene was isolated from a strain 129SV mouse genomic library. The mouse Grx1 gene consists of three exons with the first two containing the TTase protein coding region. A gene targeting vector was constructed by replacing exon 2 and some of the sequences in introns 1 and 2 with a neomycin resistance cassette. The targeting vector was linearized with NotI enzyme and then transfected into R1 embryonic stem cells. One of the 51 clones containing the targeted Grx1 allele (#89) was microinjected into blastocysts from C57BL/6 mice and the chimeric mice derived from microinjection transmitted 129 SV chromosomes into the offspring. The homozygous Grx1 KO mice were derived from breeding of heterozygous KO mice. For in vitro cataract induction, the lenses from 2 months old KO mice and wild type mice were removed and incubated in 1.5 ml TC199 medium with and without the presence of 0.5 mM H2O2 for 6 hrs in a CO2 incubator. Lens homogenate was used for TTase activity assay, and immunoblot analyses against anti–TTase monoclonal antibody and anti–protein–S–S–glutathione (PSSG) antibody. Results: The KO mice appeared to be normal and healthy at 12 months of age, but showed weak resistance to oxidative stress and developed lung tissue phenotype. The KO mice showed normal size and growth of eyes with similar wet weight of the lenses in comparison with that of the wild type. However, TTase activity was barely detectable in the lens homogenate of the KO mice. Immunoblot showed a very faint TTase protein band in comparison with the wild type. The lens contained normal levels of GSH and total soluble proteins. However, in comparison with the wild type, the lenses of KO mice showed weaker resistance to oxidative stress and formed lens opacity faster, covering most of the outer cortical regions in the lens after exposing to 0.5 mM H2O2. Both the control and the H2O2–treated lenses from KO mice displayed a positive band against anti–PSSG antibody in the region of 25–30 kDa, which was present in the H2O2 treated lens from wild type mice but not in the normal untreated group. Conclusion: A TTase KO mouse model has been established. The model provided first evidence that TTase is essential in protecting the lens from cataract formation.

Keywords: gene/expression • oxidation/oxidative or free radical damage • protein modifications–post translational 

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