Establishing and improving retroviral episome transfer of transcription factors to achieve cell fate conversion and reprogramming
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ABSTRACT: Gene transfer of distinct cocktails of transcription factors enables the targeted modification of cell fate. Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) via ectopic expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows creation of pluripotent cells with the potential to differentiate into any cell type of the body. To achieve a high degree of biosafety for future application of converted cells, technologies are required to express the required transcription factors without affecting the target cell genome. Retroviral vectors are widely employed gene transfer vehicles and can be converted into transient gene delivery vehicles by mutation of the viral integrase, thus avoiding risks associated with retroviral integration. We improved the retroviral episome transfer system by identifying the most suitable vector architecture, by enhancing episomal expression and by tuning the activity of the transcription factor Oct4. Repeated episomal transductions allowed establishment of stable, fully reversible expression levels over time. Providing proof-of-principle in the reprogramming context, we were able to generate human iPSCs following retroviral episomal transfer of Oct4 in fibroblasts stably expressing Klf4, Sox2 and c-Myc, with a high percentage of clones not carrying residually integrated vector copies. RNA obtained from human embryonic stem cells, fibroblasts and induced pluripotent stem cell lines reprogrammed with the mentioned reprogramming protocol
ORGANISM(S): Homo sapiens
SUBMITTER: Franz-Josef Müller
PROVIDER: E-GEOD-57378 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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