Project description:We discovered that mice that lack Lsd1 in hematopoietic cells were exhibited increased frequencies of CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs, but completely lacked the lin- c-Kit+ Sca-1- myeloid progenitor compartment. To determine the genes altered by Lsd1-loss, CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs from Lsd1fl/fl and Lsd1fl/fl Mx1Cre mice were FACS-purified to be analyzed by gene expression profiling.
Project description:We discovered that mice that lack Lsd1 in hematopoietic cells were exhibited increased frequencies of CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs, but completely lacked the lin- c-Kit+ Sca-1- myeloid progenitor compartment. To determine the genes altered by Lsd1-loss, CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs from Lsd1fl/fl and Lsd1fl/fl Mx1Cre mice were FACS-purified to be analyzed by gene expression profiling. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform.
Project description:We show that meteorin (Metrn) from hypoxic macrophages restrains hematopoietic stem cells (HSCs) proliferation and mobilization. In macrophages specific Metrn knockout mice, reactive oxygen species levels in HSCs were upregulated through activating phospholipase C signaling. Macrophage specific knockout mice for Metrn (Metrn-fl/fl*LysM-Cre) were generated. Transcriptome profiling (RNA-Seq) and differential gene expression analysis of bone marrow LSK (lin- sca-1+ c-kit+) cells from Metrn-fl/fl*LysM-Cre (Metrn-cKO) and Metrn-fl/fl mice was performed. Metrn cKO mice showed a regulatory role for HSPCs by macrophages.
Project description:To understand the underlying mechanism by which Alox15 gene is required by HSCs, we performed a comparative DNA microarray analysis using total RNA isolated from wild type Lin-Sca-1+c-Kit+, SELP-/- Lin-Sca-1+c-Kit+. The result was validated by quantitative real-time PCR analysis of wild type Lin-Sca-1+c-Kit+ and SELP-/- Lin-Sca-1+c-Kit+. Cancer stem cells are responsible for the initiation and maintenance of some types of cancer, and few effective target genes in these stem cells have been identified. Here we show that the selp is essential for the survival of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML). To understand the underlying mechanism through which SELP regulates the function of HSCs, the gene expression profiles between WT and Selp-/- HSCs were compared.
Project description:To understand the underlying mechanism by which the Hif1a gene is required by hematopoietic stem cells (HSCs), we performed a comparative DNA microarray analysis using total RNA isolated from wild type Lin-Sca-1+c-Kit+ cells and Hif1a-/- Lin-Sca-1+c-Kit+ cells. The result was validated by quantitative real-time PCR analysis of wild type Lin-Sca-1+c-Kit+ and Hif1a-/- Lin-Sca-1+c-Kit+ cells.
Project description:To understand the underlying mechanism by which Alox15 gene is required by HSCs, we performed a comparative DNA microarray analysis using total RNA isolated from wild type Lin-Sca-1+c-Kit+, SELP-/- Lin-Sca-1+c-Kit+. The result was validated by quantitative real-time PCR analysis of wild type Lin-Sca-1+c-Kit+ and SELP-/- Lin-Sca-1+c-Kit+. Cancer stem cells are responsible for the initiation and maintenance of some types of cancer, and few effective target genes in these stem cells have been identified. Here we show that the selp is essential for the survival of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML). To understand the underlying mechanism through which SELP regulates the function of HSCs, the gene expression profiles between WT and Selp-/- HSCs were compared. To understand the underlying mechanism through which SELP regulates the function of HSCs, the gene expression profiles between WT and Selp-/- HSCs were compared.
Project description:The study was a comparison of gene expression using RNA-seq. We analyzed the stem and progenitor cells from WT and Vav-cre+ Tet2fl/fl Flt3-ITD (T2F3) mice. We isolated stem cells LSK (lin- sca+ kit+) and granulocyte-macrophage progenitors GMP (lin- sca- kit+ fcgr+ cd34+) cells from bone marrow. Comparisons were made across genotypes WT vs. T2F3 and cell types LSK vs. GMP.
Project description:To understand the underlying mechanism by which the Hif1a gene is required by hematopoietic stem cells (HSCs), we performed a comparative DNA microarray analysis using total RNA isolated from wild type Lin-Sca-1+c-Kit+ cells and Hif1a-/- Lin-Sca-1+c-Kit+ cells. The result was validated by quantitative real-time PCR analysis of wild type Lin-Sca-1+c-Kit+ and Hif1a-/- Lin-Sca-1+c-Kit+ cells. We compared the gene profile of HSCs between WT mice and Hif1a-/- mice.
Project description:Comparison of gene expression patterns between phenotypic mouse HSCs (Lin- KIT+ SCA-1+ FLK2- CD150+ CD34-) separated into CD11A- and CD11A+ fractions. A link to our analysis of these populations can be found here (https://gexc.stanford.edu/model/1007).