Figure 1. Dual-color Bcl11b reporter strategy can reveal epigenetic mechanisms controlling T-cell lineage commitment.

(A) Overview of early T-cell development. Bcl11b turns on to silence alternate fate potentials and drive T-cell fate commitment. ETP – early thymic progenitor; DN2 – CD4^- CD8^-double negative-2A progenitor; DP – CD4⁺ CD8⁺; NK – natural killer; DC – dendritic cell. (B) Dual-allelic Bcl11b reporter cells, where two distinguishable fluorescent proteins (YFP and mCherry) are inserted non-disruptively into the same sites on the two endogenous Bcl11b loci. (C) Flow cytometry plots show cKit versus CD25 levels in CD4^-CD8^- double negative (DN) thymic progenitors (left), along with Bcl11b-YFP versus Bcl11b-mCh expression levels in the indicated DN progenitor subsets from dual Bcl11b reporter mice. Arrowheads indicate cells expressing one copy of Bcl11b. (D) Flow plots show Bcl11b-YFP versus Bcl11b-mCh levels in CD4⁺CD8⁺double positive (DP) T-cell precursors from the thymus (left), or CD4 (center) or CD8 (right) T-cells from the spleen. Results are representative of analysis of 6–8 mice from two independent experiments. See also Figure 1—figure supplement 1.

Figure 1—figure supplement 1. Experimental strategy for generating different Bcl11b reporter mouse strains.

The two Bcl11b loci were targeted in embryonic stem (ES) cells using homologous recombination, followed by drug selection using the indicated drug-resistance markers (vertical arrows). ES cells were then injected into blastocysts (horizontal arrows) to generate the indicated mice. These mice were subsequently bred to generate the appropriate two-color mice for experiments (see Materials and methods). To generate the enhancer disrupted ES cells, the dual-color tagged ES line was retargeted with a deletion construct including a selectable hygromycin resistance gene (hyg). Cells with the correct insertion were then used to generate mice.