Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Glyco-gene expression in DP thymic T cells from control and mutant mice that carry a mutation in the ligand binding domain of Notch1

ABSTRACT: Dr. Stanley's laboratory is interested in determining how O-fucose glycans function in Notch receptor signaling and in modulating the interactions of Notch receptors with their ligands. We have generated mice carrying a point mutation in the ligand binding domain of Notch1 that reduces Notch ligand binding to mutant T cells. We wish to identify Notch target genes including glycosylation genes with altered expression in DP thymic T cells from mutant and control mice. DP cells were prepared from the thymus of 3 control and 3 mutant mice at ~8 weeks of age and RNA extracted from each prep will be analysed. RNA preparations from CD4+CD8+ double positive (DP) T cells from F+2323, F+2324, F+2329 (control) and FF2330, FF2332, FF2333 (Notch1 with a point mutation to prevent fucosylation in EGF12) mice of ~8 weeks were sent to the Microarray Core (E). The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Steven Head

PROVIDER: E-GEOD-28936 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Notch1 signaling is an important regulator of cell fate in early stages of T cell development. Because Notch receptors can function redundantly, we sought an approach for inhibiting all endogenous Notch signaling in thymocytes. Upon ligand engagement, Notch receptors undergo two successive proteolytic cleavages, the second involving a Presenilin (PS) containing complex with -secretase activity that releases a small intracellular fragment of Notch. This activated form of Notch (NotchIC) translocates from the membrane to the nucleus, where it acts as a transcription factor, inducing the expression of multiple target genes . Since Presenilins are required for the activation of all four mammalian forms of Notch, we generated mice with deletions of both Presenilin1 and Presenilin2 genes, the only genes encoding Presenilin in the mouse genome. To target Notch inactivation specifically to developing T cells, we introduced Cd4-Cre to mediate Presenilin gene deletion in a tissue- and stage-specific manner. Direct target genes of Notch signaling are largely unknown, and are likely to be cell lineage and stage specific. Therefore in order to identify potential Notch target genes , we compared RNA of thymocytes at the CD4+CD8+ stage of development from H-2b transgenic 5CC7 TCR/ RAG2-deficient / Presenilin1flox/flox /PS2-/- mice, with and without Cd4-Cre. Thymocyte single cell suspensions were generated using 100 µm nylon mesh (PGC Scientifics). DP thymocytes were isolated from RAG2° control or PS1/2° 5CC7 TCR H2bb mice by magnetic bead separation on anti-CD4 coated microbeads (Miltenyi Biotec.). Total RNA was isolated using TRIzol according to manufacturer's instructions (Invitrogen). After quantification and checking for integrity, 1 µg of RNA was subjected to linear amplification. The resulting aRNA was labeled by reverse transcription via Cy5-dCTP incorporation and Cy3-dCTP incorporation. The fluorescent labeled probes were subsequently combined and hybridized on topic-defined PIQORTM Immunology Microarrays Mouse Antisense.

Dataset Information

Glyco-gene expression in DP thymic T cells from control and mutant mice that carry a mutation in the ligand binding domain of Notch1

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure