Construction of subtractive cDNA library for differentially expressed genes in rat intestinal epithelial cells exposed to high-dose radiation by suppression subtractive hybridization / 第三军医大学学报

ABSTRACT

Objective To clone and identify the differentially expressed genes of rat intestinal epithelial cell line (IEC 6) before and after exposure to high dose radiation so as to provide proof for the investigation of the molecular mechanisms in the repair of radiation damage of intestinal epithelial cells. Methods A subtractive cDNA library for differentially expressed genes was constructed by suppression subtractive hybridization (SSH) and T/A cloning technique after IEC 6 cells were exposed to radiation at the dose of 35 Gy ? ray. The expressed sequence tag (EST) library was screened by reverse Northern hybridization. Positive clones were sequenced and the similarity was searched against the DNA database in GenBank. Limited clones were identified by Northern hybridization. Results More than 2 000 white clones were harvested after the library amplification. Ninety six of them were randomly picked out for PCR amplification, and 15 positive clones which corresponded to 12 individual genes were identified by reverse Northern hybridization. These genes were involved in cell skeleton, cell stress, cell cycle control, and signal transduction, etc. In addition, a novel cDNA sequence was also obtained. Conclusion A subtractive cDNA library for differentially expressed genes in IEC 6 cells exposed to the radiation at the dose of 35 Gy ? ray has been successfully constructed with SSH and T/A clone techniques. Several positive ESTs which correspond to genes involving in cell skeleton, cell stress, cell cycle control, and signal transduction are identified. These genes may play important roles in the process of the damage and repair of the intestinal epithelial cells exposed to radiation.