The idea that females of most mammalian species have lost the capacity for oocyte production at birth has been challenged recently by the finding that juvenile and adult mouse ovaries possess mitotically active germ cells. However, the existence of female germline stem cells (FGSCs) in postnatal mammalian ovaries still remains a controversial issue among reproductive biologists and stem cell researchers. We have now established a neonatal mouse FGSC line, with normal karyotype and high telomerase activity, by immunomagnetic isolation and culture for more than 15 months. FGSCs from adult mice were isolated and cultured for more than 6 months. These FGSCs were infected with GFP virus and transplanted into ovaries of infertile mice. Transplanted cells underwent oogenesis and the mice produced offspring that had the GFP transgene. These findings contribute to basic research into oogenesis and stem cell self-renewal and open up new possibilities for use of FGSCs in biotechnology and medicine.
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gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the gene loci. We discovered that the CTCF binding site located between and genes (CBS7/9) is critical for establishing and maintaining aberrant gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior genes. Consistent with the role of the CBS7/9 boundary in locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.
. relation and complete linkage as the distance metric and clustering method, respectively. The output data were loaded into Java Tree View software to generate heatmaps (66).Statistics. Statistical differences between different experimental groups were determined by Student's t test (2 tailed). The reported values represent the mean ± SD as indicated in the figures. A P value less than 0.05 was considered statistically significant. No samples were excluded from the analysis.Study approval. All animal experimental procedures were approved by the University of Florida IACUC.
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