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Global transcriptome profiling of Oct4/Klf4/Sox2 (3Factor, 3F) + IL6 iPS clones derived from mouse embryonic fibroblasts.

ABSTRACT: We used heterokaryon cell fusion based reprogramming and identified the cytokine IL6 as a potential regulator of reprogramming to pluripotency. We generated iPS clones using the four reprogramming factors (4F) Oct4, Klf4, Sox2, and c-Myc. In addition, iPS clones were generated using only three factors (3F: Oct4, Klf4, amd Sox2) with the addition of the cytokine IL6 to reprogramming culture conditions. Global RNA-Seq of the 3F + IL6 derived iPS clones was done for comparison with 4F-derived iPS clones, mouse embryonic stem cells and mouse embryonic fibroblasts.

ORGANISM(S): Mus musculus

PROVIDER: GSE46104 | GEO | 2013/08/28

SECONDARY ACCESSION(S): PRJNA197269

REPOSITORIES: GEO

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Project description:Introducing four transcription factors, Oct4, Sox2, Klf4 and c-Myc can reprogram both human and mouse somatic cells. first, the oncogenic properties of reprogramming factors such as Klf4 and c-Myc, second, the viral integrations of transcription factors in the genome of somatic cells which may also hinder a clinical setting and finally, the low efficiency of the generation of induced pluripotent stem cells (iPS). Some recent studies overcame the first two problems, the oncogenic properties and genomic integration of the reprogramming factors by either replacing Klf4 and/or c-Myc with small molecules or using different iPS protocols such as integration-free transcription factor inductions as well as introducing proteins. However, the slow reprogramming process and low efficiency of the iPS generation still need to be improved for either clinical application or studying the reprogramming mechanism. Different approaches including adding soluble Wnt3a, and generating iPS cells in hypoxic condition also showed increased efficiency in both 4 factors (Oct4/Sox2/Klf4/c-Myc) or less factors combination without further genetic manipulation. In the current study, we show that basic fibroblast growth factor (bFGF) dramatically increased the efficiency of iPS generation and shortened the reprogramming timing of mouse embryonic fibroblast (MEF) cells in both 4 factors (4F Oct4/Sox2/Klf4/c-Myc) and 3 factors (3F Oct4/Sox2/Klf4) combination without further exogenous genetic manipulation. RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates.

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Global transcriptome profiling of Oct4/Klf4/Sox2 (3Factor, 3F) + IL6 iPS clones derived from mouse embryonic fibroblasts.

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