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| Status |
Public on Oct 13, 2016 |
| Title |
Hit and Run Protocol with Growth Factor Enhances the Generation of Mouse Induced Pluripotent Stem Cells |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
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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| Overall design |
2 samples were analyzed. Fgf2-: Mouse embryonic fibroblast culture without Fgf2 and Fgf2+: Mouse embryonic fibroblast culture without Fgf2
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| Contributor(s) |
Han DW, Araúzo-Bravo MJ, Joo JY, Greber B, Ko K, Stehling M, Schöler HR |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Nov 04, 2009 |
| Last update date |
Jan 15, 2022 |
| Contact name |
Marcos J. Araúzo-Bravo |
| E-mail(s) |
[email protected]
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| Phone |
+34 943 00 6108
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| Organization name |
Max Planck Institute for Molecular Biomedicine
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| Department |
Cell and Developmental Biology
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| Lab |
Computational Biology and Bionformatics
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| Street address |
Rogentstrasse
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| City |
Muenster |
| ZIP/Postal code |
48149 |
| Country |
Germany |
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| Platforms (1) |
| GPL6885 |
Illumina MouseRef-8 v2.0 expression beadchip |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA121287 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE18888_RAW.tar |
3.1 Mb |
(http)(custom) |
TAR |
| GSE18888_raw_data.txt.gz |
422.9 Kb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
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