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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 16, 2010 |
| Title |
Identification of genes specific to mouse primordial germ cells through dynamic global gene expression |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by array
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| Summary |
The molecular mechanisms underlying the commitment of cells to the germ cell lineage and the subsequent formation of germ cells during mammalian development remain poorly understood due to an inability to obtain sufficient amounts of cellular materials to conduct thorough in-vitro analyses. Although primordial germ cells (PGCs) have been generated and isolated from embryonic stem cells (ESCs) in vitro, concurrent expression of several cell surface receptors and transcription factors, at various levels, confounds the identification of these cells. To date, only a few genes that mark the onset of germ cell commitment to the epiblast, including tissue nonspecific alkaline phosphatase (TNAP), Blimp1, Stella, and Fragilis, have been used with some degree of success to detect PGC formation in in-vitro model systems. In light of the current literature, the generation of an unlimited supply of germ cells and gametes from pluripotent stem cells would greatly facilitate studies into reproductive development. To accomplish this, we would need to discover novel markers that are specifically expressed in germ cells. Here we identified four genes that are specifically expressed in male and female germ cells, both in vivo and in vitro, but are not expressed in somatic tissues or ESCs. Expression of these genes therefore allows us to distinguish committed germ cells from undifferentiated pluripotent cell populations, a prerequisite for the successful derivation of germ cells and gametes in vitro.
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| Overall design |
For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using RNeasy Micro kits with on-column DNase digestion as per the manufacturer's instructions. Integrity of RNA samples was quality-checked using a 2100 Bioanalyzer (Agilent). If 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 hrs 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 hrs 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 the iScan reader and accompanying software.
44 samples were analyzed.
ESCm: OG2 male Embryonic Stem Cell, +/+ Oct4-GFP ES cells, 1 biological rep
11.5: 11.5 embryonic day PGC (Primordial Germ Cell), 2 biological reps
12.5F: 12.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
13.5F: 13.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
14.5F: 14.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
15.5F: 15.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
16.5F: 16.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
17.5F: 17.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
18.5F: 18.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
12.5M: 12.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
13.5M: 13.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
14.5M: 14.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
15.5M: 15.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
16.5M: 16.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
17.5M: 17.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
18.5M: 18.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
8M: 8 day after birth male PGC (Primordial Germ Cell), 2 biological reps
10M: 10 day after birth male PGC (Primordial Germ Cell), 2 biological reps
12M: 12 day after birth male PGC (Primordial Germ Cell), 2 biological reps
14M: 14 day after birth male PGC (Primordial Germ Cell), 1 biological rep
16M: 16 day after birth male PGC (Primordial Germ Cell), 2 biological reps
18M: 18 day after birth male PGC (Primordial Germ Cell), 2 biological reps
20M: 20 day after birth male PGC (Primordial Germ Cell), 2 biological reps
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| Contributor(s) |
Sabour D, Araúzo-Bravo MJ, Hübner K, Ko K, Greber B, Gentile L, Stehling M, Schöler HR |
| Citation(s) |
20940145 |
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| Submission date |
Jul 29, 2010 |
| 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 (44)
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| Relations |
| BioProject |
PRJNA131231 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE23322_RAW.tar |
3.1 Mb |
(http)(custom) |
TAR |
| GSE23322_non-normalized.txt.gz |
7.3 Mb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
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