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| Status |
Public on Jun 22, 2023 |
| Title |
Defining the Sox2-independent reprogramming steps to pluripotency |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
Several Sox family members, small molecules, and inhibitors of the TGF-β pathway can replace exogenous Sox2 during the generation
of induced pluripotent stem cells (iPSCs), suggesting that SOX2 is dispensable for initiation of reprogramming. However,
the time point at which the endogenous Sox2 locus is activated in these conditions was not thoroughly evaluated. To identify the Sox2-independent
reprogramming steps, we excluded Sox2 from the reprogramming cocktail and used Sox2-deficient mouse embryonic fibroblasts.
We show that reprogramming is initiated in the absence of SOX2 as shown by formation of pre-iPSC colonies that have undergone a
mesenchymal-to-epithelial transition and that have activated early reprogramming markers.
However, these iPSC-like intermediates cannot progress through the final stabilization phase of reprogramming in the absence of SOX2.
Overall, our results provide a better understanding of the molecular events underlying the induction of pluripotency and of the role that
SOX2 plays in the process.
Both pLV-tetO-E1A-2A-Cherry and pLV-tetO-Cherry lentiviral constructs were cloned in house and defective-lentiviral particles were
produced as previously described (Han et al. 2013).
Next, 5x10 4 ESCs containing a tetracycline-inducible transactivator (irtTA-VP16-GBD, Anastassiadis et al. 2002) plus an Oct4-GFP reporter
transgene (Yoshimizu et al. 1999) were transduced twice using 20 µl of replication-defective lentiviral particles plus polybrene (4 µg/ml, Sigma).
Cells were washed 24 h after transduction and subsequently induced with doxycycline (1 µg/ml, Sigma) and dexamethasone (10-7 M, Sigma).
Samples were harvested at several time-points, 0, 3, 6, 9, 12, 24, and 48 hours after induction,
and used for microarray analysis as previously reported (Tapia et al. 2012).
Cells transduced with pLV-tetO-Cherry and induced for 48 h were used as control.
RNA wasprocessed using the RNeasy micro and mini kits with on-column DNase digestion of QIAGEN as per the manufacturer’s instructions.
Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When possible,
300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA Amplification Kit, Ambion),
which involved synthesis of T7-linked double-stranded cDNA and 14 hr of in-vitro transcription incorporating biotin-labeled nucleotides.
Purified and labeled cRNA was then quality checked on a 2100 Bioanalyser and hybridized as biological or technical duplicates for 18 hr onto
MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed,
the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader and accompanying software.
Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina).
Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model.
Variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the
lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab.
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| Overall design |
16 samples were analyzed
0h, Mouse Embryonic Stem Cells (ESC) after 0 hours of E1A induction, 2 replicates
3h, Mouse Embryonic Stem Cells (ESC) after 3 hours of E1A induction, 2 replicates
6h, Mouse Embryonic Stem Cells (ESC) after 6 hours of E1A induction, 2 replicates
9h, Mouse Embryonic Stem Cells (ESC) after 9 hours of E1A induction, 2 replicates
12h, Mouse Embryonic Stem Cells (ESC) after 12 hours of E1A induction, 2 replicates
24h, Mouse Embryonic Stem Cells (ESC) after 24 hours of E1A induction, 2 replicates
48h, Mouse Embryonic Stem Cells (ESC) after 48 hours of E1A induction, 2 replicates
48hc, Mouse Embryonic Stem Cells (ESC) after 48 hours of E1A induction, cherry control, 2 replicates
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| Contributor(s) |
Marthaler AG, Adachi K, Tiemann U, Wu G, Araúzo-Bravo MJ, Sabour D, Velychko S, Kleiter I, Schöler HR, Tapia N |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| Submission date |
Jul 14, 2014 |
| Last update date |
Jun 22, 2023 |
| Contact name |
Marcos J. Araúzo-Bravo |
| E-mail(s) |
mararabra@yahoo.co.uk
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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 (16)
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| Relations |
| BioProject |
PRJNA255213 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE59372_RAW.tar |
3.1 Mb |
(http)(custom) |
TAR |
| GSE59372_non-normalized.txt.gz |
2.6 Mb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
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