Generation of transducible versions of transcription factors Oct4 and Sox2.
The transcription factors Oct4 and Sox2 are two of the main regulators of pluripotency in embryonic stem cells. Since the importance of non-genetic modification is continually increasing, particularly for therapeutic application of manipulated cells, the aim of the present study was to generate cell-permeant Oct4 and Sox2 proteins for the direct cellular delivery of active proteins. Protein transduction allowing cellular manipulation to circumvent genetic modification of target cells has recently been developed. We present a new expression vector system, pSESAME, that facilitates the generation of transducible proteins. Using pSESAME, both Oct4 and Sox2 were genetically fused with a TAT protein transduction domain that promotes cellular penetration. The recombinant purified Oct4 and Sox2 fusion proteins display DNA-binding properties comparable to their endogenous counterparts, and exhibit cellular entry and the ability to modulate the transcriptional machinery maintaining pluripotency of mouse embryonic stem cells. In a rescue assay we demonstrate that transducible Oct4 and Sox2 fusion proteins can compensate knockdown of Pou5f1 and Sox2, respectively. This study provides powerful tools for the modulation of stem cell properties without genetic interference.
Bosnali
Manal
M
Institute of Reconstructive Neurobiology, Stem Cell Engineering Group, University of Bonn, Life & Brain Center and Hertie Foundation, Sigmund-Freud Str. 25, Bonn, Germany.
Edenhofer
Frank
F
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D053595:Embryonic Stem Cells / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D024002:HMGB Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D024002:HMGB Proteins; D050814:Octamer Transcription Factor-3; D034741:RNA, Small Interfering; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D014157:Transcription Factors; D004247:DNA