Project description:Expression profiling of Rag2-deficient Ets1++ and Rag2-deficient Ets1-- mature NK cells and WT bone marrow progenitors, WT T cells, and WT Pro B cells
Project description:Expression profiling of Rag2-deficient Ets1++ and Rag2-deficient Ets1-- mature NK cells and WT bone marrow progenitors, WT T cells, and WT Pro B cells WT Hematopoietic progenitors, CD4 T cells, Pro B cells, and WT and Ets1-deficient NK cells were FACs sorted. RNA was subsequently extracted, labelled, and hybridized to Affymetrix microarrays. The goal if this experiment was to identify Ets1 dependent genes in NK cells
Project description:T-cell development is predicated on the successful rearrangement of the T-cell receptor (TCR) gene loci, which encode for antigen-specific receptors that enable an immune response. Recombination Activating Gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T-cell development. Here, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCRb chain in mediating b-selection during human T-cell development in vitro. In stark contrast to what is seen in mice, human RAG2-KO T-lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRb rearrangements. Nevertheless, RAG2-KO DPs retrovirally-transduced to ectopically express a rearranged TCRb chain showed increased survival and proliferation as compared to control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRb-transduced and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide new insights as to the distinct requirement for the TCRb chain during human T-cell development.