Gene expression changes in Itpr triple conditional knockout T cell progenitors

Calicum plays critical roles in developing T cells, promoting survival, proliferation, and differentiation at multiple stages of maturation. Calcium release from intracellular stores due to T cell receptor and pre-T cell receptor signaling is thought to require the inositol triphosphate receptors (Itpr), however the requirement for these receptors has not been investigated. We used microarrays to examine gene expression differences between control and Itpr deficient T cell progenitors. We used microarrays to identify changes in the transcriptional programme of developing T cells prior to acquisition of a mature T cell receptor and identified several developmentally important genes altered in the absence of Itpr. Thymocyte progenitors at DN4 or ISP were harvested for RNA extraction from three independent control Itpr1/Itpr2/Itpr3 triple homozygous floxed (CTL) or Tie2-Cre Itpr1/Itpr2/Itrp3 triple homozygous floxed (KO) thymii, pooled, and hybridized on Affymetrix microarrarys. To obtain a homogenous population of thymocyte progenitors, we FACS purified cells and collect RNA from only DN4 or ISP T cells. ISP T cells were defined as CD4- CD8+ CD24+ CD3-. DN4 T cells were defined as CD45+ CD24+ CD3- CD4- CD8- CD25- CD44- CD19- B220- gdTCR- Ter119- Mac1- Gr1-. Thymocytes were harvested from neonatal mice, ranging from 7 to 10 days of age and lacked any signs of malignancy or disease.