Purchase this article with an account.
S.H. Liu, A.L. Bowers, A.J. Michels, A.J. Jun, J.D. Gottsch; Differentially Expressed Genes in Human Corneal Endothelium Using Suppression Subtractive Hybridization . Invest. Ophthalmol. Vis. Sci. 2003;44(13):850.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To generate a cDNA library enriched in transcripts that are predominantly expressed in corneal endothelium by suppression subtractive hybridization (SSH) and differential screening. Methods: Full-length cDNAs were synthesized from corneal endothelium isolated from human donor tissue. Normal endothelial cDNAs (tester) were subtracted against stromal and epithelial cDNAs (driver) to eliminate genes common to epithelial cells and keratocytes. Screening with tester and driver probes were used to identify differentially expressed cDNA clones. Clones that failed to hybridize to driver probes, but strongly hybridized to the subtracted tester probes, were sequenced. Differential expression of obtained genes was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Results: The sequence of 263 cDNAs from a subtracted endothelial library was determined. Sequencing demonstrated 184 known genes. Sixteen corresponded to ESTs and 4 were novel. Twenty-three of the known genes and two of the ESTs were detected more than once. The majority of known genes are related to matrix metabolism and cell-matrix interaction (matrix metalloproteinase, tissue inhibitor of metalloproteinase, galectin and matrix Gla protein), to antioxidants and proteins conferring protection against toxic stress and apoptosis (superoxide dismutase, gluthione S-transferase, carbon anhydrase, peroxiredoxin and apoptosis inhibitor), and to intracellular signaling (chitinase 3-like, serum amyloid A1 and A2, FBJ murine osteosarcoma viral oncogene homolog, and interferon induced transmembrane protein). These known genes had their highest expression in the corneal endothelium when compared to other tissues and analyzed by RT-PCR. A novel clone was studied which had weak homology with a member of the apoptosis inhibitor family. This new gene was expressed predominantly in corneal endothelium with absent or minimal expression in corneal epithelium, stroma and other non-ocular tissues. Conclusion: Subtractive hybridization and differential screening of human corneal endothelium has identified a number of genes that demonstrate preferential expression for this tissue. These genes may be responsible for the specialized functions of the corneal endothelium
This PDF is available to Subscribers Only