Identification of mouse blastocyst genes that are downregulated by double-stranded RNA-mediated knockdown of Oct-4 expression

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Abstract

Oct-4 is an essential transcription factor involved in the differentiation of the inner cell mass (ICM) in mouse blastocysts, and is thought to be the pluripotent gene of embryonic stem cells. However, downstream genes of Oct-4 and the mechanism by which it regulates the transcription machinery remain unclear. Here, we specifically knocked down Oct-4 gene expression in mouse blastocysts by double-stranded RNA (dsRNA) interference. A recently developed method, the annealing control primer (ACP) technique, was then used to identify the downstream genes of Oct-4. By using 120 arbitrary ACP, 10 clones were found to be differentially expressed in the knocked down embryos and the cloned genes were analyzed by DNA sequencing and BLAST searching. Quantitative real time reverse transcription (RT)-polymerase chain reaction (PCR) confirmed that the expression of these genes is altered by Oct-4 knockdown. Of the 10 genes, 8 (Atp6ap2, GK003, Ddb1, hRscp, Dppa1, Dpp3, Sap18, and Rent1) were downregulated and 2 (Rps14 and ETIF2B) were upregulated in Oct-4 dsRNA-injected blastocysts. One of the downregulated genes is developmental pluripotency associated-1 (Dppa1), which has already been identified as being an Oct-4 downstream gene. Two other genes, Rent1 and Sap18, were found to be Oct-4 downstream genes for the first time. The genes identified here will provide insights into the roles played by Oct-4 during embryonic development.

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