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TATκ Fusion Protein of OCT-3/4 and KLF-4: Stable Mixed Population Cell Lines Capable of Delivering Fusion Proteins to Target Cells

Fazlina Nordin, Gee Jun Tye, Joop Gaken and Farzin Farzaneh

A rapidly growing body of evidence has shown that enforced expression of a small panel of genes, including Oct-3/4, KLF4, Sox2 and c-Myc, can induce the reprogramming of previously differentiated cells, to generate induced Pluripotent Stem Cells (iPSCs). However, the generation of iPSCs by genetic modifications raises the risk of malignant transformation. Therefore, efficient in vitro reprogramming of differentiated cells, without irreversible genetic modification is highly desirable. The improved trans-activator of transcription kappa (TATκ), a synthetic TAT-HIV, confers the ability to deliver several proteins into target cells, thus, making it a potential alternative delivery mechanism for transcription factors or therapeutic agents. Using Green Fluorescence Protein (GFP) and Apoptin as model proteins, we have recently described a strategy to generate cell lines that secrete proteins carrying a modified HIV-TAT Protein Transduction Domain (PTD) for subsequent protein transduction mediated uptake by target cells. Using this strategy we have generated 293T cells secreting the pluripotent factors Oct-3/4 or KLF4 in fusion with TATκ. Oct-3/4 and KLF4 were detected in the culture medium of the transduced 293T cell and uptake of Oct-3/4 by haematopoietic cell lines JURKAT and FDCP-1 was confirmed by Western blot analysis. KLF4 was present in the culture medium of the producer 293T cell but we are still unable to demonstrate uptake by target cells. Based on results obtained, we hope that these stable mix population cell lines can play an important role in generation of iPSCs for therapeutic purposes.