Construction of a Plasmid Containing Attachment Site (attB) Sequence Related to ФC31 Integrase Next to GFP cDNA with Murine Oct-4 Promoter

Ghorbani Jarghouyeh R *, Khazaie Y , Dormiani K , Forouzanfar M , Karbalaie KH , Karamali F , Nematollahi M ,


Objective: In order to study the Oct-4 regulation and its function in the differentiation process, we have designated to clone its related promoter upstream of EGFP as a gene marker.Oct-4 is a transcription factor of the POU family. This protein is critically involved in the self-renewal of differentiated embryonic stem cells and also is a transcription factor used to create induced pluripotent stem cells, demonstrating its capacity to induce an embryonic stem cell-like state. As such, it is used as a marker for undifferentiated cells. Materials and Methods: OCT4 promoter was cloned using murine genome and placed near to EGFP gene as a marker gene for further analysis. Moreover the recognition sites for integrase were put near to the constructed sequences for efficient insertion into the target genomes. Results: Sequence analysis and transfection data confirmed that accuracy of cloned promoter which EGFP showed a clear fluoresceny upon tranfection into the mouse embryoninc stem cells. Conclusion: Co-transfecting of this plasmid and a vector expressing integrase cDNA, into the murine stem cell line (Royan B1), we obtained numerous transformant cell colonies expressing EGFP under regulation of this promoter