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| Status |
Public on Jul 11, 2013 |
| Title |
Reprogramming of two somatic nuclei in the same ooplasm leads to pluripotent embryonic stem cells |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
We provide proof that the oocyte is able to reprogram two somatic nuclei at once and therefore is not limited in its reprogramming capacity to a single nucleus. Mouse ooplasts transplanted with 2 somatic cell nuclei simultaneously (double nuclear transfer) support preimplantation development and the derivation of tetraploid NT embryonic stem cells (tNT-ES cells). These cells are unique in that they embody the first case of cloned ES cells containing two reprogrammed somatic genomes at the same time and exhibit characteristics of true pluripotency.
By assessing allelic expression of Oct4 in normal and double nucleus transplanted embryos throughout development we demonstrate that oocyte mediated reprogramming is embarked with a probabilistic component and that one reprogrammed genome can dominate the other during preimplantation development even if merged into the same nucleus. Animal handling and cell recovery: Oocytes from mice of the B6C3F1 strain were used for SCNT and ICSI. Cumulus cells (for SCNT) and sperm (for ICSI) were either from a B6C3F1 or OG2 donor the latter expressing an Oct4 promoter-driven GFP transgene. Additionally cells from pure C56Bl/6 or C3H mice were retrieved for SCNT. Female mice were sacrificed by cervical dislocation 14 h after hCG injection and oocytes retrieved from oviduct ampullae. Oocytes and cumulus cells were handled in Hepes-buffered CZB (HCZB) medium. Double nuclear transfer ICSI and embryo culture: Micromanipulations and embryo culture were performed at 30°C (room temperature). For SCNT oocytes were first enucleated (spindle-chromosome complex removed) before transfer of one two or three cell nuclei with a micropipette driven by a piezo actuator under Nomarski optics. The reconstructed oocytes were activated for 6 h in Calcium-free alpha-MEM supplemented with 10 mM SrCl2 and 5 micro-g mL-1 cytochalasin B. Intracytoplasmic sperm injection (ICSI) of one two or four into intact oocytes was used as the fertilization control condition using sperm from 8-week-old mice of B6C3F1 or OG2 strains. Embryos were placed in embryo culture medium and cultured at 37°C under 5% CO2. The activated oocytes were cultured in alpha-MEM at 37°C and under 5% CO2 until use. On day 5 after activation SCNT and ICSI blastocysts were processed for ESC derivation. Analysis of allelic expression of Oct4: RNA from single embryos was extracted using the ZR-RNA MicroPrep kit (Zymo Research). Complementary DNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Invitrogen) according to manufacturers instruction. A nested PCR was performed to amplify part of the Oct4 cDNA containing a BamH1 sensitive SNP. Primer sequences and PCR conditions are given in the supplementary methods. The PCR product was digested for 8h using 100 units of BamH1-HF (NEB) subsequently subjected to agarose gel electrophoreses and the results grouped into 3 categories (mainly C57Bl/6 balanced mainly C3H) according to the observed band patterns. Global transcriptome analysis by microarray: RNA samples to be analyzed by 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 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto a MouseRef-8 v2 expression BeadChip (Illumina) following the manufacturer 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 exclusively hybridized as biological replicates. Microarray data processing: The 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 Background correction performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics was performed with in-house developed functions in Matlab.
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| Overall design |
8 samples were analyzed.
tNT#1: tetraploid nuclear transfer, p17, double sedimentation;
tNT#2: tetraploid nuclear transfer, p19, double sedimentation;
tNT#3: tetraploid nuclear transfer, p25, double sedimentation;
tNT#4: tetraploid nuclear transfer, p29, double sedimentation;
tNT#5: tetraploid nuclear transfer, p18, double sedimentation;
tNT#6: tetraploid nuclear transfer, p25, double sedimentation;
NT#1: nuclear transfer, double sedimentation;
NT#2: nuclear transfer, double sedimentation;
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| Contributor(s) |
Pfeiffer MJ, Esteves TC, Balbach ST, Araúzo-Bravo MJ, Stehling M, Jauch A, Houghton F, Boiani M |
| Citation(s) |
23922292 |
| |
| Submission date |
Mar 13, 2012 |
| Last update date |
Jan 15, 2022 |
| Contact name |
Marcos J. Araúzo-Bravo |
| E-mail(s) |
[email protected]
|
| 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
|
| Street address |
Rogentstrasse
|
| 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 (8)
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| GSM894347 |
tetraploid nuclear transfer #1 rep1 |
| GSM894348 |
tetraploid nuclear transfer #2 rep1 |
| GSM894349 |
tetraploid nuclear transfer #3 rep1 |
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| Relations |
| BioProject |
PRJNA153491 |
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