ABSTRACT
CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Lymphoma , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Lymphoma/drug therapy , Lymphoma/genetics , Lymphoma/pathology , Mice , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-myc/metabolism , SulfonamidesABSTRACT
BACKGROUND: The quality of RBC components is influenced by collection, processing and storage conditions. Regulations require that whole blood (WB) units be refrigerated within 8 hours and processed into RBCs within 24 hours of collection. Overnight room temperature hold of WB has logistical advantages, but the effect on RBC quality has not been fully investigated. RBC additive solutions were compared for their ability to provide improved quality of RBCs prepared from WB held at room temperature for 24 hours. STUDY DESIGN AND METHODS: Leukocyte-reduced RBCs were prepared from WB held at 20°C on cooling plates for 24 hours prior to processing. RBCs were stored in additive solutions, SAG-M (control), Erythrosol-4, and PAGGSM, under standard blood banking conditions and sampled during 49 days of storage. Stored RBCs were evaluated for RBC shape and microparticle (MP) accumulation using flow cytometry. Osmotic fragility, adhesion of RBCs to endothelium under shear stress conditions (0.5 dyne/cm(2) ), and routine RBC quality parameters were assessed. RESULTS: RBCs stored in Erythrosol-4 and PAGGSM had decreased cell size, reduced osmotic fragility, and decreased accumulation of glycophorin A-positive MPs and annexin V-binding MPs compared with RBCs stored in SAG-M. RBCs stored in erythrosol-4 had increased adherence to endothelium at days 42 and 49 compared with RBCs stored in SAG-M or PAGGSM. CONCLUSION: RBCs stored in PAGGSM or Erythrosol-4 had improved retention of RBC membrane and osmotic resilience. The development of new additive solutions may offer improved quality of RBC components prepared from WB held overnight at room temperature.