P-39: Cloning and Expressing an HA-Tagged OCT4-B1 Construct in Stem and Tumor Cell Lines (Pages: 57-57)

Khalediyan B *, Farashahi E , Asadi H , Fathi F , Kabir Salmani M , Mowla SJ ,


Objective: OCT4 is a master regulator of self-renewal and involved in regulating pluripotency in embryonic stem cells (ESCs). In addition to the previously described spliced variants of the gene (e.g. OCT4A and OCT4B), we have recently identified a novel variant of the gene, designated as OCT4-B1. The expression of OCT4-A is limited to ESCs and ECCs, but not expressed in other non-pluripotent cells. While OCT4-B is expressed at low levels, when detected, OCT4B1 is expressed highly in ESCs and ECCs and its expression decreases with the onset of differentiation. OCT4-B1 is highly upregulated in various tumors and cell-lines, where it acts as an anti-apoptotic factor. Materials and Methods: We have designed specific siRNAs to suppress OCT4-B1 expression in stem and tumor cell lines. We have also constructed an HA-tagged OCT4B1, which generates an N-terminal tagged protein, detectable by HA antibody. The transfected cells then were lysed and the expression of HA-OCT4-B1 transcript and protein detected by means of real-time PCR and Western Blotting. Results: Our data revealed that interfering with the expression of OCT4-B1 causes profound changes in the morphology and cell cycle distribution of the cells. Furthermore, Western blotting revealed a protein product with a molecular marker corresponding to OCT4B variant and a truncated protein with a molecular size corresponding to OCT4-B1. Conclusion: Our data demonstrated a functional role for OCT4-B1 in apoptosis and tumorigenesis. Our data also revealed that OCT4-B1 could act as a precursor to OCT4B protein and that it could also generate a truncated OCT4-B1 protein.