Construction of subtracted cDNA library for differentially expressed genes associated with chronic optic nerve injury / 第二军医大学学报

ABSTRACT

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.