Bisulfite genomic sequencing of microdissected cells

Kerjean, Antoine; Vieillefond, Annick; Thiounn, Nicolas; Sibony, Mathilde; Jeanpierre, Marc; Jouannet, Pierre

doi:10.1093/nar/29.21.e106

Cited by 32 publications

(17 citation statements)

References 10 publications

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“…Two promoter sequences also exhibited differential methylation, at the Hccs and the 5730596B20Rik loci. We used a technique published for microdissected cells (Kerjean et al 2001) for bisulfite conversion, digesting DNA from the same brain and spermatogenic cell samples used for the HELP assay overnight, denaturing the DNA using heat and NaOH, and embedding it in agarose beads. The DNA was then treated with sodium bisulfite for 4 h at 50°C and then washed in TE pH 8.0, followed by desulfonation with 0.2 M NaOH.…”

Section: Discussionmentioning

confidence: 99%

Comparative isoschizomer profiling of cytosine methylation: The HELP assay

et al. 2006

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The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.

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Section: Discussionmentioning

confidence: 99%

Comparative isoschizomer profiling of cytosine methylation: The HELP assay

et al. 2006

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“…p15 MSP on sorted cell populations was carried out as described (29). After sorting, the cells were embedded in agarose beads and lysed overnight with 0.5 mol/L EDTA (pH 8.0)/2 mg/mL proteinase K at 50jC.…”

Section: Methodsmentioning

confidence: 99%

Hematopoietic Stem Cells Are Not the Direct Target of Spontaneous Leukemic Transformation in p18INK4C-Null Reconstituted Mice

Yuan

Yu²,

Boyer³

et al. 2006

Cancer Research

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Cell cycle inhibitors are important regulators in normal tissue regeneration and disruption of the regulators are involved in cancer development. Our recent study showed that the absence of the CDK inhibitor p18 INK4C (p18) enhances self-renewal of normal hematopoietic stem cell (HSC) in vivo, whereas previous studies by others showed an increased incidence of leukemogenesis in older p18-null mice. Here, we have examined potential leukemogenesis during experimentally induced regeneration of HSC in the absence of p18 in order to gauge the relation between these two processes. Reconstituted mice with p18-deficient HSCs under the condition of repetitive proliferative stress (serial transplantation) were followed for >3 years. T cell leukemia from the p18À/À origin was recapitulated 24 months after secondary transplantation. However, no myeloid leukemia was found in the recipients. The T cell leukemia-initiating cells (mainly in a CD3 lo cell subset) did not share the same immunophenotype with normal HSCs and, in fact, the function of HSCs was significantly compromised with decreased abundance in the leukemic mice. Furthermore, we found that the p15 or p16 gene promoters were frequently methylated in the leukemic cells but not in HSCs. Our present study argues against the possibility of overgrowth of p18-null HSCs leading to a leukemic phenotype. The data also support the notion that p18 has an independent role in T cell maintenance such that CD3 + CD8 + cells, unlike HSCs, are more accessible to leukemogenic transformation after the loss of p18. (Cancer Res 2006; 66(1): 343-51)

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“…9 Special care was taken to avoid contamination from granulosa cells. DNA methylation analysis of specific differentially methylated regions (DMRs) of H19, Mest/ Peg1 and Igf2R was performed on independent pools of 5 -8 fg oocytes by denaturing high-performance liquid chromatography (DHPLC) analysis as recently reported.…”

Section: Methodsmentioning

confidence: 99%

In vitro follicular growth affects oocyte imprinting establishment in mice

et al. 2003

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In vitro folliculogenesis of cryopreserved ovarian tissue could be an effective method for insuring fertility for patients who receive gonadotoxic treatment. Although several culture systems have been described for growing female gametes in vitro, the production of competent oocytes for further development remains a considerable challenge. The purpose of our study was to determine whether maternal primary imprinting progresses normally during mouse oocyte growth in vitro. We analysed the DNA methylation status of differentially methylated regions of the imprinted genes H19, Mest/Peg1 and Igf2R using fully grown germinal vesicle-stage oocytes (fg oocytes) produced by in vitro folliculogenesis from early preantral follicles. When compared to fg oocytes removal from control females, we observed after in vitro development, a loss of methylation at the Igf2R locus in six out of seven independent experiments and Mest/Peg1 locus (one out of seven), and a gain of methylation at the H19 locus (one out of seven). These results provide insight into the dysregulation of the process of primary imprinting during oocyte growth in vitro and highlight the need for effective new biomarkers to identify complete nuclear reprogramming competence after in vitro folliculogenesis.

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Bisulfite genomic sequencing of microdissected cells

Cited by 32 publications

References 10 publications

Comparative isoschizomer profiling of cytosine methylation: The HELP assay

Comparative isoschizomer profiling of cytosine methylation: The HELP assay

Hematopoietic Stem Cells Are Not the Direct Target of Spontaneous Leukemic Transformation in p18INK4C-Null Reconstituted Mice

In vitro follicular growth affects oocyte imprinting establishment in mice

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