Widespread and efficient deletion of IFNαβR in vivo following tamoxifen activation of Cre-ERT2.

Abstract

<p>(A) LCMV immune UBC-Cre-ERT2⁺IFNαβR^fl/fl and UBC-Cre-ERT2^-IFNαβR^fl/fl mice were injected with tamoxifen, and the indicated analyses were carried out. (B) The efficiency of deletion at the DNA level was assessed by PCR analysis of genomic DNA extracted from the indicated tissues; M = markers; Splns = splenocytes; LN = lymph nodes; x = empty lane. 1144bp = floxed allele, 309bp = deleted allele. (C & D) The efficiency of Cre activation in T cells of LCMV-immune mice was determined. Cre⁺ or Cre^- IFNαβR^f/fzsGreen^+/wt mice were injected with tamoxifen and, two weeks later, splenocytes were harvested and incubated with each of the four indicated peptides, and virus-specific T cells were identified using standard intracellular cytokine staining for IFNγ. zsGreen expression also was evaluated. The red numbers indicate zsGreen⁺ cells as a percentage of all CD8⁺ T cells (No Peptide groups) or of virus-specific (i.e, IFNγ⁺) cells (peptide-stimulated groups). (C) Gated on CD8⁺ T cells and (D) gated on CD4⁺ T cells. (E) The efficiency with which Cre activation resulted in ablation of IFNαβR biological function was determined, by measuring Stat 1 phosphorylation after <i>in vitro</i> incubation with IFNβ. ZsGreen+CD8⁺ T cells from Cre⁺ mice were compared to CD8⁺ T cells from Cre^- animals. Grey histograms = isotype control antibody. Mouse numbers: C & D, n = 6–7; E, n = 4.</p