Identification of putative downstream genes of Oct-4 by suppression-subtractive hybridization.

As a step toward understanding how toti/pluripotence is maintained by Oct-4, we have first constructed a cell model with differentially expressed Oct-4 in embryonic stem cells, and then used suppression-subtractive hybridization (SSH) method to identify the downstream genes of Oct-4. Among the 384 clones we screened, 40 clones were detected as differentially expressed genes with colony hybridization, and 13 clones were confirmed as the putative downstream genes of Oct-4 by Northern blot analysis. Sequencing showed 12 different genes, 8 known genes (Oct-4, Rex-1, Sox-2, Creatine kinase B, Makorin 1, Importin beta, Histone H2A.Z, Ribosomal protein S7) and 4 new genes. Except Oct-4 and Rex-1, the other genes have not been reported to be regulated by Oct-4. These results showed that SSH provides a very efficient means to identify the downstream genes of transcription factor. Some known genes identified may provide new insight of the function of Oct-4 in stem cells. Copyright 2001 Academic Press.