Screening for endocrine exophthalmos-associated genes/cDNA fragments / 生物医学工程学杂志

ABSTRACT

This study was conducted to screen the endocrine exophthalmos-associated genes or cDNA fragments and provide a basis for exploring the pathogenesis. The cDNA from the thyroid tissues of patients with hyperthyroidism and endocrine exophthalmos (HT&EE) was used as tester cDNA, and that from the thyroid tissues of patients with HT but free from EE was used as driver cDNA. The subtractive PCR products between tester and driver cDNA were obtained through two cycles of subtraction hybridization and two cycles of PCR by using suppression subtractive hybridization, and then were inserted into pT-Adv, a cloning vector. The ligated DNAs were transformed into E. coli DH5alpha competent cells and incubated for proper blue/white color development. Forty-eight white colonies were randomly picked and their inserts were colony PCR amplified. The PCR product from one of the colonies contained two inserts; the others contained single insert, having a size of 0.2 kb to 2 kb. The inserts of transformants were arrayed on nylon membrane. After cDNA/Rsa I digestion the thyroid tissues of patients with HT, and of patients with HT&EE, were labeled with digoxigenin; the nylon membranes were then hybridized respectively with the two cDNA probes for a high throughput screening for positive clones. The clones which were hybridized with the cDNA probe of HE&EE patients but not hybridized with the probe of the HT patients or showed only faint signal of hybridization, were chosen as positive clones and their inserts were candidates for the endocrine exophthalmos genes or cDNA fragments. About 50% of the clones were confirmed as positive clones. The cDNA fragments in the positive clones were the endocrine exophthalmos-associated genes or cDNA fragments. Endocrine exophthalmos