Résumé
Objective: To clone, identify and analyze subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve compression in cats. Methods: The constructed cDNA libraries were amplified by E. coli transformation with calcium chloride and were subjected to blue and white screening. Three hundred positive bacterial clones were randomly chosen from each library and were identified by colony PCR. The positive clones were sequenced to screen for the differentially expressed genes. The identified sequences were then analyzed for homology using Blast program against the DNA database bank of Japanese through internet. Real-time PCR was performed to verify the expression of the 4 differential clones. Results: One thousand positive clones were identified by PCR and 674 ESTs were obtained by seqeuncing, with length ranging from 200 to 800bp. Results from Blast analysis revealed 14, 20, 23 and 19 homolog genes in 4-w forward, 4-w reverse, 8-w forward and 8-w reverse subtractive libraries, respectively. These genes fell into several functional groups, such as energy and metabolism, ion transport, gene transcription, signal transduction, cellular injury and repair, and MHC molecules. Result of real-time PCR verified that the 4 clones were differentially expressed. Conclusion: The constructed subtractive cDNA libraries lay an experimental basis for further identification and study of the differentially expressed genes related to chronic optic nerve injury.
Résumé
Objective: To construct subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury in cats. Methods: Fifteen adult cats were randomly divided into 3 groups (n=5): control group, 4-w compression group and 8-w compression group. The chronic optic nerve injury was produced by an inflatable balloon implanted under the optic chiasm. The total RNA was prepared from optic nerves of each group by TRIzol method. Double-stranded cDNA was produced by SMART PCR cDNA synthesis protocol. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes in the optic nerves after 4-w and 8-w compression. The cDNA fragments were directly inserted into T/A cloning vector to establish the subtractive library,followed by amplification of the libraries through E. coli transformation with calcium chloride and screening of blue and white clones. Three hundred positive bacterial clones were randomly picked in each library and identified by colony PCR. Results: Analysis of the white clones by PCR showed that 80% clones contained 200-800 bp inserts in each library. Conclusion: Four subtractive cDNA libraries of differentially expressed genes associated with chronic optic nerve injury have been successfully constructed by SSH and T/A cloning techniques,which lays a solid foundation for screening and cloning specific differentially expressed genes associated with chronic optic nerve injury.