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Genome-wide analysis of total skin gene expression from presenilin (1&2)-null (PSmut), RBPjk-null (RBPjmut), Presenilin (1&2)-RBPjk-null (PSRmut) and wild-type (WT) littermates

ABSTRACT: Analysis of differential gene expression of total skin from different Notch signaling pathway mutants (specifically PSmut, RBPjmut, and PSRmut). Preselin1&2 are the core components of g-secretase enzyme which essential for the Notch receptor activation. RBPjk is the DNA-binding partner of the Notch Intracellular Domain. The hypothesis tested in the present study was RBPjmut skin would resemble the wild-type skin at the transcriptional level, since RBPjmut mice exhibit milder phenotype than PSmut or PSRmut mice.

ORGANISM(S): Mus musculus

PROVIDER: GSE77887 | GEO | 2016/02/29

SECONDARY ACCESSION(S): PRJNA311954

REPOSITORIES: GEO

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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

Genome-wide analysis of total skin gene expression from presenilin (1&2)-null (PSmut), RBPjk-null (RBPjmut), Presenilin (1&2)-RBPjk-null (PSRmut) and wild-type (WT) littermates

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