Mus musculus
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PROVIDER: PRJNA181955 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: To investigate the effect of Tet1 depletion on global DNA methylation, we performed whole-genome bisulfite sequencing (WGBS). Methods: Starting with as little as 1400-5251 manually micro-dissected PGCs, we used an ultra-low input method, Tn5mC-seq. Results: We generated 945 million reads for Tet1Gt/Gt PGCs and 302 million reads for wild-type PGCs. We obtained 14-16 million CpG sites per genotype at 1.76-2.66x genome coverage, which enables a comprehensive view of genome-wide DNA methylation patterns in E13.5 PGCs. PGCs are almost completely unmethylated genome-wide. Loss of Tet1 led to a subtle increase of methylation level in various genomic elements including promoters, exons, introns and repetitive elements in Tet1Gt/Gt PGCs (p<0.01). Local analysis identified 4,337 differentially methylated regions (DMRs) between Tet1-/- PGCs and wild-type cells. These DMRs are associated with 5,261 genes, among which 271 genes also exhibited differential gene expression and enriched for the cell cycle pathway (FDR=0.02). Conclusions: This result revealed that demethylation of certain set of cell cycle genes is largely abolished in the Tet1-/- PGCs. Genome-wide methylation profiles of primordial germ cells derived from the wild type (WT) and Tet1-null female embryos at E13.5 were generated by whole genome bisulfite sequencing using Illumina Hiseq.
2012-11-20 | E-GEOD-41912 | biostudies-arrayexpress
Project description:Transcriptome profiling of wild type primordial germ cells (PGC) by RNAseq
| PRJNA178509 | ENA
Project description: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. 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
2010-11-16 | E-GEOD-23322 | biostudies-arrayexpress
Project description:Genomic imprinting is an allele-specific gene expression system important for mammalian development and function. The molecular basis of genomic imprinting is allele-specific DNA methylation 2. While it is well known that the de novo DNA methyltransferases Dnmt3a/b are responsible for the establishment of genomic imprinting, how the methylation mark is erased during primordial germ cell (PGC) reprogramming remains a mystery. Here we report that Tet1 plays a critical role in the erasure of genomic imprinting. We show that despite their identical genotype, progenies derived from mating between Tet1-KO males and wild-type females exhibit a number of variable phenotypes including placental, fetal and postnatal growth defects, and early embryonic lethality. These defects are, at least in part, caused by the dysregulation of imprinted genes, such as Peg10 and Peg3, which exhibit aberrant hypermethylation in the paternal allele of differential methylated regions (DMRs). RNA-seq reveals extensive dysregulation of imprinted genes in the next generation due to paternal functional loss of Tet1. Genome-wide DNA methylation analysis of E13.5 PGCs and sperm derived from Tet1-KO mice reveals hypermethylation of DMRs of imprinted genes in sperm, which can be traced back to PGCs. Dynamics of methylation change in Tet1-affected sites suggested that Tet1 swipes remaining methylation including imprinted genes at late reprogramming stage. We also revealed that Tet1play a role in paternal imprinting erasure in females germline. Thus, our study establishes a critical function for Tet1 in the erasure of genomic imprinting. Genome-wide DNA methylation analysis of E13.5 PGCs from control and Tet1-KO mice
2013-12-02 | E-GEOD-49762 | biostudies-arrayexpress
Project description:Genomic imprinting is an allele-specific gene expression system important for mammalian development and function. The molecular basis of genomic imprinting is allele-specific DNA methylation 2. While it is well known that the de novo DNA methyltransferases Dnmt3a/b are responsible for the establishment of genomic imprinting, how the methylation mark is erased during primordial germ cell (PGC) reprogramming remains a mystery. Here we report that Tet1 plays a critical role in the erasure of genomic imprinting. We show that despite their identical genotype, progenies derived from mating between Tet1-KO males and wild-type females exhibit a number of variable phenotypes including placental, fetal and postnatal growth defects, and early embryonic lethality. These defects are, at least in part, caused by the dysregulation of imprinted genes, such as Peg10 and Peg3, which exhibit aberrant hypermethylation in the paternal allele of differential methylated regions (DMRs). RNA-seq reveals extensive dysregulation of imprinted genes in the next generation due to paternal functional loss of Tet1. Genome-wide DNA methylation analysis of E13.5 PGCs and sperm derived from Tet1-KO mice reveals hypermethylation of DMRs of imprinted genes in sperm, which can be traced back to PGCs. Dynamics of methylation change in Tet1-affected sites suggested that Tet1 swipes remaining methylation including imprinted genes at late reprogramming stage. We also revealed that Tet1play a role in paternal imprinting erasure in females germline. Thus, our study establishes a critical function for Tet1 in the erasure of genomic imprinting. Genome-wide DNA methylation analysis of sperm derived from control and Tet1-KO mice
2013-12-02 | E-GEOD-49763 | biostudies-arrayexpress
Project description:Genomic imprinting is an allele-specific gene expression system important for mammalian development and function. The molecular basis of genomic imprinting is allele-specific DNA methylation 2. While it is well known that the de novo DNA methyltransferases Dnmt3a/b are responsible for the establishment of genomic imprinting, how the methylation mark is erased during primordial germ cell (PGC) reprogramming remains a mystery. Here we report that Tet1 plays a critical role in the erasure of genomic imprinting. We show that despite their identical genotype, progenies derived from mating between Tet1-KO males and wild-type females exhibit a number of variable phenotypes including placental, fetal and postnatal growth defects, and early embryonic lethality. These defects are, at least in part, caused by the dysregulation of imprinted genes, such as Peg10 and Peg3, which exhibit aberrant hypermethylation in the paternal allele of differential methylated regions (DMRs). RNA-seq reveals extensive dysregulation of imprinted genes in the next generation due to paternal functional loss of Tet1. Genome-wide DNA methylation analysis of E13.5 PGCs and sperm derived from Tet1-KO mice reveals hypermethylation of DMRs of imprinted genes in sperm, which can be traced back to PGCs. Dynamics of methylation change in Tet1-affected sites suggested that Tet1 swipes remaining methylation including imprinted genes at late reprogramming stage. We also revealed that Tet1play a role in paternal imprinting erasure in females germline. Thus, our study establishes a critical function for Tet1 in the erasure of genomic imprinting. Gene expression analysis of E9.5 embryos
2013-12-02 | E-GEOD-49761 | biostudies-arrayexpress
Project description:The requirement for primordial germ cells (PGCs) during sexual differentiation is variable among vertebrates. It has been shown that in zebrafish complete loss of PGCs in the embryos causes exclusive male development. Further, transplantation of a single PGC into a germline deficient zebrafish embryos generates male exclusively, suggesting PGC number might be important for the ovarian fate.To explore how PGC number might regulate sexual development in zebrafish, we experimentally manipulated its number by injecting dnd MO into embryos to generate fish containing a spectrum of PGC number. The experiment was designed to compare transcriptomes between the developing trunk regions of wild type and dnd morphants at different developmental stages.
2015-05-08 | GSE57046 | GEO
Project description:The requirement for primordial germ cells (PGCs) during sexual differentiation is variable among vertebrates. It has been shown that in zebrafish complete loss of PGCs in the embryos causes exclusive male development. Further, transplantation of a single PGC into a germline deficient zebrafish embryos generates male exclusively, suggesting PGC number might be important for the ovarian fate.To explore how PGC number might regulate sexual development in zebrafish, we experimentally manipulated its number by injecting dnd MO into embryos to generate fish containing a spectrum of PGC number. The experiment was designed to compare transcriptomes between the developing trunk regions of wild type and dnd morphants at different developmental stages. The dnd MO was microinjected into one cell stage embryos to generate zebrafish with a range of PGC numbers. Transcriptomes of developing trunk regions of wild type and dnd morphants at 14 dpf and 22 dpf were analyzed.
2015-05-08 | E-GEOD-57046 | biostudies-arrayexpress
Project description:Genomic imprinting is an allele-specific gene expression system important for mammalian development and function. The molecular basis of genomic imprinting is allele-specific DNA methylation 2. While it is well known that the de novo DNA methyltransferases Dnmt3a/b are responsible for the establishment of genomic imprinting, how the methylation mark is erased during primordial germ cell (PGC) reprogramming remains a mystery. Here we report that Tet1 plays a critical role in the erasure of genomic imprinting. We show that despite their identical genotype, progenies derived from mating between Tet1-KO males and wild-type females exhibit a number of variable phenotypes including placental, fetal and postnatal growth defects, and early embryonic lethality. These defects are, at least in part, caused by the dysregulation of imprinted genes, such as Peg10 and Peg3, which exhibit aberrant hypermethylation in the paternal allele of differential methylated regions (DMRs). RNA-seq reveals extensive dysregulation of imprinted genes in the next generation due to paternal functional loss of Tet1. Genome-wide DNA methylation analysis of E13.5 PGCs and sperm derived from Tet1-KO mice reveals hypermethylation of DMRs of imprinted genes in sperm, which can be traced back to PGCs. Dynamics of methylation change in Tet1-affected sites suggested that Tet1 swipes remaining methylation including imprinted genes at late reprogramming stage. We also revealed that Tet1play a role in paternal imprinting erasure in females germline. Thus, our study establishes a critical function for Tet1 in the erasure of genomic imprinting.
2013-12-02 | GSE49763 | GEO
Project description:Genomic imprinting is an allele-specific gene expression system important for mammalian development and function. The molecular basis of genomic imprinting is allele-specific DNA methylation 2. While it is well known that the de novo DNA methyltransferases Dnmt3a/b are responsible for the establishment of genomic imprinting, how the methylation mark is erased during primordial germ cell (PGC) reprogramming remains a mystery. Here we report that Tet1 plays a critical role in the erasure of genomic imprinting. We show that despite their identical genotype, progenies derived from mating between Tet1-KO males and wild-type females exhibit a number of variable phenotypes including placental, fetal and postnatal growth defects, and early embryonic lethality. These defects are, at least in part, caused by the dysregulation of imprinted genes, such as Peg10 and Peg3, which exhibit aberrant hypermethylation in the paternal allele of differential methylated regions (DMRs). RNA-seq reveals extensive dysregulation of imprinted genes in the next generation due to paternal functional loss of Tet1. Genome-wide DNA methylation analysis of E13.5 PGCs and sperm derived from Tet1-KO mice reveals hypermethylation of DMRs of imprinted genes in sperm, which can be traced back to PGCs. Dynamics of methylation change in Tet1-affected sites suggested that Tet1 swipes remaining methylation including imprinted genes at late reprogramming stage. We also revealed that Tet1play a role in paternal imprinting erasure in females germline. Thus, our study establishes a critical function for Tet1 in the erasure of genomic imprinting.
2013-12-02 | GSE49762 | GEO