Figure 12.

Scheme of generation, maintenance, genetic modification and differentiation of Cas9-Hoxb8 cells. Lineage-negative cells from the bone marrow of Cas9 transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline (Dox)-regulated form of the transcription factor Hoxb8 and a puromycin (Puro) selection cassette. Cas9-Hoxb8 cells could be kept for weeks in a non-differentiated state by culture in the presence of mSCF, huIL-11, huFlt3L, mIL-3, Dox, and Puro. Genetic modifications such as overexpression or CRISPR/Cas9-mediated knockout could be introduced by viral transduction. Successfully targeted cells were selected based on fluorescence-activated cell sorting. The longevity of Cas9-Hoxb8 cells provided the opportunity to knockout multiple genes by consecutive genetic manipulations. For differentiation into macrophages or dendritic cells (DCs), mSCF, huIL-11, huFlt3L, mIL-3, Dox, and Puro were replaced with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF).