Unknown,Transcriptomics,Genomics,Proteomics

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Sox17-transgenic hematopoietic stem cell microarray

ABSTRACT: The transcription factor SOX17 is expressed by fetal, but not adult hematoipoietic stem cells (HSCs), and is required for the maintenance of fetal and neonatal, but not adult, HSCs. In the current study we show that ectopic expression of Sox17 in adult HSCs and transiently reconstituting multipotent progenitors was sufficient to confer increased self-renewal potential and the expression of fetal HSC genes including fetal HSC surface markers. To assess the acute effects of ectopic Sox17 expression on global gene expression in adult HSCs, we performed microarray analysis to compare the gene expression profile of adult Sox17-trangenic and control HSCs after short induction of Sox17-transgene expression. Total RNA were isolated from 5 independent, freshly isolated aliquots of 10,000 HSCs isolated from 8-week old Sox17-transgenic ((tetO)7CMVSox17-IRES-NucEGFP;B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae/J double transgenic) or littermate control mice that were treated with doxycycline for 5 days to induce transgene expression. Purified RNA was reverse transcribed and amplified using the WT-Ovation™ Pico RNA Amplification system (NuGEN Technologies) following the manufacturer’s instructions. Sense strand cDNA was generated using WT-Ovation™ Exon Module (NuGEN), then fragmented and labeled using the FL-Ovation™ cDNA Biotin Module V2 (NuGEN). 2.5µg of labeled cDNA were hybridized to Affymetrix Mouse Gene ST 1.0 microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Shenghui He

PROVIDER: E-GEOD-30445 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Sox17-transgenic hematopoietic stem cell microarray

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