Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC
- 517 Downloads
It has been shown that human and murine fibroblasts can be reprogrammed by ectopic expression of transcription factors using viral vectors. For the purpose of human therapeutic applications, the integration of viral transgenes into the genome is unlikely to be accepted. We therefore produced recombinant transcription factor proteins in E. coli (OCT4, SOX2, c-MYC and KLF4) carrying the cell penetrating TAT domain from HIV1. The purified proteins were able to enter into mammalian cells when added to tissue culture medium but appeared not to translocate to the nucleus. Further investigation indicated that most of the protein was tied up in the endosomes and was unavailable for reprogramming. Once this problem has been solved it seems likely that protein reprogramming will be the method of choice for clinical applications.
KeywordsInduced pluripotent stem cells (iPSCs) TAT peptide Reprogramming Transcription factors Gene expression
We are grateful to Lisa Dailey for providing the 6X0/S-luc luciferase reporter plasmid. We thank Zhan Wang (Wake Forest Institute for Regenerative Medicine) for his help in experiments. C. Pan was supported by CHINA SCHOLARSHIP COUNCIL. This work was supported by a grant from National Institutes of Health (NIH) (R21 RR025408).
- 16.Ambrosetti DC, Scholer HR, Dailey L, Basilico C (2000) Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer. J Biol Chem 275:23387–23397. doi: 10.1074/jbc.M000932200 CrossRefPubMedGoogle Scholar
- 17.Jin LH, Bahn JH, Eum WS, Kwon HY, Jang SH, Han KH, Kang TC, Won MH, Kang JH, Cho SW, Park J, Choi SY (2001) Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells. Free Radic Biol Med 31:1509–1519. doi: 10.1016/S0891-5849(01)00734-1 CrossRefPubMedGoogle Scholar
- 18.Park J, Kim KA, Ryu JY, Choi EY, Lee KS, Choi SY (2000) Generation and characterization of cell-permeable green fluorescent protein mediated by the basic domain of human immunodeficiency virus type 1 Tat. J Microbiol Biotechnol 10:797–804Google Scholar
- 20.Yoshikawa T, Sugita T, Mukai Y, Yamanada N, Nagano K, Nabeshi H, Yoshioka Y, Nakagawa S, Abe Y, Kamada H, Tsunoda S, Tsutsumi Y (2008) Organelle-targeted delivery of biological macromolecules using the protein transduction domain: potential applications for Peptide aptamer delivery into the nucleus. J Mol Biol 380:777–782. doi: 10.1016/j.jmb.2008.05.047 CrossRefPubMedGoogle Scholar