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Beschreibung

Various methods utilizing viruses, DNA, RNA, miRNA and protein have been developed to generate integration-free induced pluripotent stem cells (iPSCs). Disadvantages to existing methods include: (1) low reprogramming efficiency (i.e. DNA and protein), (2) a lengthy requirement for negative selection and subcloing steps to remove persistent traces of the virus (i.e. Sendai virus)1, (3) daily transfections of cells using four synthetic mRNAs over a 14 day period (i.e. mRNA based)2, and (4) the inability to directly assess transfection or viral transduction efficiency of the introduced reprogramming factors.Our Simplicon OKSG-cMyc TagRFP RNA uses a safe and efficient method to generate integration- and virus-free human iPSCs using a single transfection step. The technology utilizes a single self-replicating Venezuelian equine encephalitis (VEE) RNA species that expresses the reprogramming factors (RF) ORFs3 (OKSG-cMyc, Oct4, Klf4, Sox2, Glis1 and cMyc) along with a red fluorescent protein (TagRFP). The Simplicon RNA replicon is a synthetic polycistronic VEE-RF RNA that is capable of self-replicating in a limited number of cell divisions. The OKSG-cMyc transgenes are especially useful for iPSCs generation from somatic cells that are more difficult to reprogram (i.e. slower proliferating cells or aged somatic cells) while the TagRFP provides a rapid assessment of transfection efficiency. Presence of the TagRFP transgene also allows for optimization of the transfection conditions in hard- to- transfect somatic or primary cells.