Dissecting the interaction between Gata1s and miR-125b with a miR-125b-mimic shRNA library
We have previously described that overexpression of miR-125b in fetal liver cells (FLCs) harboring the Gata1s mutation (Gata1s-FLCs) leads to expansion of megakaryocyte progenitors in vitro and ML-DS-like leukemia upon transplantation into C57BL/6J recipients in vivo. We aimed to decipher which are the targets of miR-125b whose loss synergizes with Gata1s. For that purpose, we have designed a library of shRNAs against 220 predicted targets of miR-125b.  Upon transduction of Gata1s-FLCs with the pool of shRNAs, we observed an initial depletion followed by an expansion after 10 days in culture, indicating the outgrowth of certain shRNAs. We performed next generation sequencing (NGS)-based shRNA quantification 4 days and 30 days post-transduction.