Figure 2.

Framework for experimental perturbation studies to define functions of PU.1 in early thymic development. Stages of cells are as in Figure 1 (DN3: primarily DN3a). Exogenous PU.1 is added by retroviral transduction. Note the dependence of the PU.1 functions tested upon the timing of the experimental perturbation. Endogenous PU.1 can be deleted by Cas9 plus single-chain guide RNAs (sgRNA) against Spi1, or by introducing Cre into cells with floxed Spi1 alleles. PU.1 can also be neutralized by adding a dominant negative construct. The DN2a-DN2b interval is accessible to experimental perturbation. The approximate developmental stage represented by the Scid.adh.2C2 cell line (see text) is also shown [Schematic adapted from Ungerbäck et al. (85)].