TET2_IDH1_TALEN

This study is a major part of a preliminary investigation into the suitablilty of Transcription Activator-Like Effector Nucleases (TALENs), specifically designed for the TET2 and IDH1 loci, in editing primary mouse heamatopoietic cells. Previous optimisation assays, in our hands, have shown these same TALENs (supplied by a commercial vendor, Cellectis) to be ~1-2% efficient at genome editing in mouse ES cells. Due to the nature of mouse ES cell culture, single cell colonies can be selcted and the genome editing potential of these reagents assessed by screening individually derived colonies by standard Sanger sequencing. In vitro culture of heamatopoietic cells does not readily permit derivation of single cell clones and as such bulk sequencing to assess the editing potential is required. In a wider context, these optimisation/proof of principle experiments will seek to complement the use of genetically sensitised mouse knock-in/kncock-out models of leukaemogenic mutations, as already identified by next generation sequencing of individual Acute Myeloid Leukaemia genomes. A major aim of our groups' work is to understand how the complement of leukaemogenic mutations act synergistically in vivo to drive leukaemogenesis. Future studies will utilise these TALEN reagents (and others) to genome edit mouse heamatopoietic cells derived from mice which are genetically and conditionally sensitised to accute myeloid leukaemia (AML). Post TALEN genome editing, these cells will be transplanted into syngeneic recipients and the onset of AML monitored using techniques established in the lab.