Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse T-cell thymocytes and bone marrow-derived macrophages dervied from Upf2fl/fl;LysMCre and Upf2fl/fl;LckCre mice to test the importance of nonsense-mediated mRNA decay on global transcription

ABSTRACT: Knockdown experiments in transformed human cells, suggested that nonsense-mediated mRNA decay (NMD) components had extensive effects on the regulation of a significant fraction of the transcriptome In order to test the importance of NMD on global transcription we subjected BMDMs and T-cell thymocytes derived from Upf2fl/fl;LysMCre and Upf2fl/fl;LckCre mice, respectively, to microarray-based gene expression profiling. Both these cell types display strong, if not complete, reduction of NMD as assayed by transfection with reporter constructs or sequencing of Tcrb transcripts. Upf2¯/¯ BMDMs displayed significant changes in the expression of 182 genes (234 probe sets) of which 179 were upregulated.

ORGANISM(S): Mus musculus

SUBMITTER: Charlotte Schjerling

PROVIDER: E-MEXP-1371 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements.

Weischenfeldt Joachim J Damgaard Inge I Bryder David D Theilgaard-Mönch Kim K Thoren Lina A LA Nielsen Finn Cilius FC Jacobsen Sten Eirik W SE Nerlov Claus C Porse Bo Torben BT

Genes & development 20080501 10

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance process that eliminates mRNAs containing premature termination codons (PTCs). NMD has been hypothesized to impact on several aspects of cellular function; however, its importance in the context of a mammalian organism has not been addressed in detail. Here we use mouse genetics to demonstrate that hematopoietic-specific deletion of Upf2, a core NMD factor, led to the rapid, complete, and lasting cell-autonomous extinction ...[more]

PMID: 18483223

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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.

Dataset Information

Transcription profiling of mouse T-cell thymocytes and bone marrow-derived macrophages dervied from Upf2fl/fl;LysMCre and Upf2fl/fl;LckCre mice to test the importance of nonsense-mediated mRNA decay on global transcription

Publications

NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements.

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