Project description:CRISPR/Cas9-mediated gene targeting of Sox9 from embryonic mammary progenitors cell line eMPC1. Part of a study to assess the effect of Sox9 ablation on embryonic mammary progenitor cell fate and function.
Project description:RNA from MMTV-Cre;Sox9flox/flox mouse mammary glands were compared to RNA from MMTV-Cre;Sox9+/flox glands. Results indicate that Sox9 regulates several genes that impact ductal morphogenesis in the mammary gland.
Project description:RNA from MMTV-Cre;Sox9flox/flox mouse mammary glands were compared to RNA from MMTV-Cre;Sox9+/flox glands. Results indicate that Sox9 regulates several genes that impact ductal morphogenesis in the mammary gland. The portion of the fourth mammary gland that is proximal to the intra-mammary gland lymph nodes was dissected from four 5-week-old MMTV-Cre;Sox9flox/flox females and four MMTV-Cre;Sox9+/flox females of the same age. Total RNA from each gland was extracted and hybridized to separate Affymetrix Gene 1.0 ST chips.
Project description:Embryonic mammary cells are a unique population comprised of undifferentiated, highly plastic progenitor cells that create normal mammary tissues. The mammary gland continues to develop after birth from descendants of embryonic mammary cells. Here, we establish cell lines from mouse mammary organs, immediately after they formed during prenatal development, to facilitate studies of primitive mammary cells, which are difficult to isolate in sufficient quantities for use in functional experiments. We show that some lines can be induced to secrete milk, a distinguishing feature of mammary epithelial cells. Targeted deletion of Sox9, from one clone, decreases the ability to respond to lactogenic stimuli, consistent with a previously identified role for Sox9 in regulating luminal progenitor function. Sox9 ablation also leads to alterations in 3D morphology and downregulation of Zeb1, a key epithelial-mesenchymal transition regulator. Prenatal mammary cell lines are an invaluable resource to study regulation of mammary progenitor cell biology and development.
Project description:RNA-seq of embryonic mammary progenitor cell lines. Embryonic mammary progenitor cell (eMPC) clones were derived from E12.5-stage mammary organs. After microdissection, mammary primordial 3 was cultured on thick basement membrane extract for 4 weeks. After enzymatic dissociation eMPC swere plated and expanded in 2D culture to obtain a pool of eMPCs. eMPC cells were subject to single cell sorting using FACS. Clones were expanded as single-cell derived clones (eMPC1, 2, etc.) to create the eighteen single cell-derived clones described in this study.
Project description:To define the repertoire of Sox9-dependent genes that contribute to the regulation of chondrogenesis, we generated Sox9-3'enhanced green fluorescent protein (EGFP) knock-in mice (Sox9-3'EGFP) and Sox9-EGFP/EGFP null chimeras. EGFP-positive cells of Sox9-3'EGFP knock-in and Sox9-EGFP/EGFP null chimeric embryos harvested from limb buds at embryonic day 12.5 were sorted using a FACSAria flow cytometer (Becton-Dickinson). Total RNA of sorted cells was extracted using the RNeasy Mini Kit (QIAGEN) and amplified according to the instructions provided by Affymetrix. Microarray analysis using the Affymetrix Mouse Genome 430 2.0 Array was performed according to the manufacturer's instructions.
Project description:The study aims to show that Sox9+ Chd1+ mouse embryonic lung progenitors can be isolated and expanded long-term in 3D culture while maintaining their multipotency. in vitro cultured Sox9+ Chd1+ lung progenitors transcriptionally resemble their in vivo counterparts and show significant difference from adult lung epithelial (Cdh1+ and EpCAM+) and non-epithelial (Cdh1- and EpCAM-) cells