Loss of STAT5 is incompatible with NK-cell viability. (A) PCR genotyping of MACS-purified splenic NK cells of Stat5^f/f and Stat5^f/fNcr1-iCreTg mice. Four mice per genotype were pooled. (B) Real-time PCR analysis of Stat5a and Stat5b mRNA levels of FACS-sorted splenic CD3⁻DX5⁺ NK cells. Ten mice per genotype were pooled. (C) Histograms show percentage and mean fluorescence intensity of various NK-cell markers on CD3⁻NKP46⁺ splenocytes of the indicated genotypes. (D) MACS-purified splenic NK cells were cultured under IL2. After 10 days of culture, only those cells that expressed Stat5 expanded, as indicated by the lack of a Stat5 deletion band on PCR analysis (left panel). Flow cytometry confirmed the NK-cell nature of these cells (right panel). Dot plot indicates CD3⁻DX5⁺ cells. Four mice per genotype were pooled. (E) MACS-purified splenic NK cells from Stat5^f/f mice were cultured under IL2 (4 mice per genotype were pooled) and infected with Ad/Cre-GFP (indicated as Stat5^Δ) or mock-infected (indicated as Stat5^f/f). Those cells that received the empty vector tolerated the expression of Ad/GFP, whereas those that had received Ad/Cre-GFP expressed the Cre recombinase and declined (left panel). PCR genotyping of the cells confirmed the deletion of Stat5 in Ad/Cre-GFP–infected NK cells (right panel). Data are representative of at least 2 independent experiments. FT, flow-through.