Project description:This lineage tracing experiment focused on studying the transcriptome of cardiac myeloid cells derived from BM HSCs post-MI. We performed Left coronary artery ligation (LAD) in a tamoxifen-inducible in vivo HSC-lineage tracing mouse model which allows HSC-progeny lineage tracing via Tomato expression (Fgd5-CreERT2 tdTomato; PMID: 30561324). This was followed by a 2-day treatment with either vehicle (DMSO) or 4-oxo-RA. On day 3 post-MI, hearts were extracted, and tdTomato-positive and tdTomato-negative myeloid cells (CD11b+) were isolated from the myocardium. scRNA-seq was performed.
Project description:RNA sequencing of FACS sorted microglia isolated from brain tissue of WT, Hexb-tdTomato, Hexb-CreERT2 and Hexb-KO mice was performed to test for potential differences in gene expression caused by the knock-in of tdTomato and CreERT2 into the Hexb locus.
Project description:RNA sequencing of FACS sorted microglia isolated from brain tissue of WT, Hexb-tdTomato, Hexb-CreERT2 and Hexb-KO mice was performed to test for potential differences in gene expression caused by the knock-in of tdTomato and CreERT2 into the Hexb locus.
Project description:RNA sequencing of FACS sorted microglia isolated from brain tissue of WT, Hexb-tdTomato, Hexb-CreERT2 and Hexb-KO mice was performed to test for potential differences in gene expression caused by the knock-in of tdTomato and CreERT2 into the Hexb locus.
Project description:Smooth muscle cells (SMCs) undergo phenotypic switching during atherosclerosis and give rise to a large fraction of plaque cells. To investigate the role of hypoxia-inducible factor 1α (HIF1α) signaling in SMC behaviour during atherogenesis, we used mice in which Hif1a is conditionally deleted in lineage-traced SMCs (Myh11-CreERT2; Rosa26-tdTomato; Hif1a-floxed). Cre recombination was induced with tamoxifen at 6 weeks of age. Atherosclerosis was induced by a single tail-vein injection of rAAV8-PCSK9 at 9 weeks of age followed by 21 weeks of high-fat diet feeding. Aortic arches were pooled by genotype from SMC-specific Hif1a knockout (Hif1aSMC-KO, n=8) and wild-type control (Hif1aWT, n=6) male mice, enzymatically dissociated, and viable cells (Draq5+/DAPI–) isolated by fluorescence-activated cell sorting. Single-cell transcriptomes were generated using the 10x Genomics Chromium Next GEM Single Cell 3′ v3.1 platform and sequenced on an Illumina HiSeq 4000. Reads were aligned to the mm10 reference transcriptome supplemented with the tdTomato transgene sequence using Cell Ranger. The dataset enables comparison of cell-type composition and of SMC-derived cell transcriptional states between genotypes, with a focus on hypoxia- and stress-response pathways underlying SMC phenotypic modulation. Only male mice were studied because the Myh11-CreERT2 transgene is Y-linked.
Project description:All current smooth muscle cell (SMC) restricted Cre mice recombine floxed alleles in vascular and visceral SMCs. We generated a tamoxifen-inducible CreERT2 mouse, Itga8-CreERT2, and compared its activity to the widely used Myh11-CreERT2 mouse. Both CreERT2 mice showed similar activity in vascular SMCs; however, Itga8-CreERT2 displayed low activity in visceral SMC-containing tissues (e.g., intestine). Myh11-CreERT2 (but not Itga8-CreERT2) mice exhibited high levels of CreERT2 protein, tamoxifen-independent activity, and an altered transcriptome. Whereas Myh11-CreERT2-mediated knockout of Srf resulted in a lethal intestinal phenotype, loss of Srf with Itga8-CreERT2 (SrfItga8) revealed viable mice with attenuated vascular SMC contractile gene expression, but no evidence of intestinal pathology. Male and female SrfItga8 mice presented with vascular contractile incompetence; however, only male SrfItga8 mice showed systemic changes in blood pressure. These results establish the Itga8-CreERT2 mouse as an alternative to existing SMC Cre strains where gene loss may result in visceral myopathies that obfuscate accurate phenotyping in vascular SMCs.
Project description:We used microfluidic single cell RNA-seq on adult isolated CC10-CreERT2 (negative) integrin beta4(pos) cells lung epithelial cells in order to determine the transcriptional profile of this putative progenitor population. CC10-CreERT2 / tdTomato (negative) integrin beta4(pos) cells were isolated by FACS, as were Krt5-CreERT2 / tdTomato (positive) cells. These cells were pooled and loaded onto the Fluidigm C1 device.