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Construction of human corneal endothelial cDNA library and identification of novel active genes
Rieko Sakai1, Tadatoshi Kinouchi, Shoko Kawamoto
1Department of Ophthalmology, Jichi Medical School, Kawachi-gun, Tochigi, Japan.
Purpose:
To describe genes expressed in human corneal endothelial cells and identify novel genes.
Methods:
Sixteen human donor corneas that had no history of corneal disease, infection, or intraocular surgery were used within 7 days of death. Total RNA was extracted from corneal endothelial cells with attached Descemet membranes. A 3'-directed cDNA library was constructed from mRNA by using a pUC19-based primer. These sequences were compared with each other to determine their frequency and were searched against GenBank for identification. To identify novel specific and abundant transcript genes in corneal endothelial cells, the novel genes were compared with an expressed sequence tag database, the expected sequence extended, and 5' rapid amplification of cDNA ends-polymerase chain reaction cloning performed.
Results:
The human corneal endothelial cDNA library showed that the most abundant transcript was prostaglandin D2 synthase. The remaining transcript genes that were present in abundance consisted of lactate dehydrogenase-A, gene signature (GS) 3582, which is a novel gene without a known function, and matrix Gla protein. The full-length sequence of GS3582 showed similarity to genes obtained in ovary and TESTIS.
Conclusions:
A human corneal endothelial cDNA library was constructed. An expression profile of corneal endothelium provides probes to monitor physiologic and pathologic conditions of this tissue in terms of gene expression.
Insights
Researchers created a human corneal endothelial cell gene library, identifying prostaglandin D2 synthase as the most abundant transcript and discovering a novel gene, GS3582. This provides a profile for monitoring corneal health.
Area of Science:
- Ophthalmology
- Molecular Biology
- Genetics
Background:
- The human corneal endothelium is crucial for maintaining corneal clarity.
- Understanding gene expression in corneal endothelial cells is vital for diagnosing and treating eye conditions.
Purpose of the Study:
- To characterize the genes expressed in human corneal endothelial cells.
- To identify novel genes within this cell type.
Main Methods:
- Construction of a 3'-directed complementary DNA (cDNA) library from human corneal endothelial cells.
- Sequencing and comparison of cDNA library transcripts against GenBank and expressed sequence tag databases.
- Utilizing 5' rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) for novel gene identification and characterization.
Main Results:
- Prostaglandin D2 synthase was identified as the most abundant transcript in the corneal endothelial cDNA library.
- Other abundant transcripts included lactate dehydrogenase-A and matrix Gla protein.
- A novel gene, GS3582, with unknown function and similarity to genes in ovary and testis, was identified.
Conclusions:
- A comprehensive cDNA library of human corneal endothelial cells was successfully constructed.
- The generated gene expression profile serves as a valuable tool for assessing physiological and pathological states of the cornea.
- This research lays the groundwork for future studies on corneal endothelial cell function and disease.