December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Creation of Human Eye cDNA Libraries for Differential Screening and Yeast Two-Hybrid Analysis
Author Affiliations & Notes
  • MW Sharp
    University of Alberta Edmonton AB Canada
    Medical Genetics
  • JS Friedman
    University of Alberta Edmonton AB Canada
  • V Raymond
    Laboratoire d'endocrinologie moléculaire Centre de recherche du Centre Hospitalier de l'Université Laval Quebec City PQ Canada
  • MA Walter
    Ophthalmology and Medical Genetics
    University of Alberta Edmonton AB Canada
  • Footnotes
    Commercial Relationships   M.W. Sharp, None; J.S. Friedman, None; V. Raymond, None; M.A. Walter, None.
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 2437. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      MW Sharp, JS Friedman, V Raymond, MA Walter; Creation of Human Eye cDNA Libraries for Differential Screening and Yeast Two-Hybrid Analysis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2437.

      Download citation file:

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

  • Supplements

Abstract: : Purpose: Glaucoma can be caused by anterior chamber malformations and/or misfunctions in the developing and adult eye. To discover genes involved in glaucoma, we are creating cDNA libraries from human eye-tissues. Applications of these cDNA libraries include identifying differentially-expressed ocular genes and discovering proteins that interact with known glaucoma genes. Methods: 50 human eyes were dissected from fetuses (9-13 weeks gestation) and total RNA was extracted. The mRNA was then isolated from the fetal eye total RNA with a poly-A RNA extraction kit and was used as the starting material for cDNA synthesis. With protocol alterations to the Cytotrap library construction system (Stratagene), the cDNA was size-fractionated and cloned into a modified two-hybrid prey vector. The pMyr vector was mutagenisized to include recombinational cloning sites facilitating the shuttling of the cDNA library or specific cDNA clones into other vectors while maintaining the frame of the cDNA inserts. PCR was initially performed on the cDNA library to determine the presence or absence of glaucoma-associated genes. Results: The human fetal eye cDNA library includes approximately 3.8 x 104 primary colony forming units (cfu). The PCR analysis indicated the presence of FOXC1, Oculoglycan, and PITX2 transcripts as expected. Human adult trabecular meshwork tissue collection, dissection and the total RNA extraction have already been completed. We intend to create and test a human adult trabecular meshwork cDNA library in the same fashion. Conclusion: These eye-specific cDNA libraries will be excellent and unique tools for use in identifying genes involved in ocular devlopment and function. Two-hybrid analyses of the fetal eye cDNA library using known glaucoma genes as bait are in progress.

Keywords: 476 molecular biology • 420 genetics 

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.