Generation of Dicer^c/c mice and embryonic fibroblasts. (A) Dicer exons 15–17 flanked with loxP sites in targeted ES cells. Cre-excision generates a conditional allele (middle) and a null allele deleted for Dicer exons 15–17 (bottom). (B) Southern analysis of F1 mice indicates transmission of both Dicer-modified alleles. Marker band positions are shown at left. (C) Phenotype of Dicer wild-type (WT) and mutant (MT) mice at E7 (darkfield image). Bar, 250 μM. (D) Strategy for creating and analyzing Dicer-ablated MEFs.

Dicer ablation induces DNA damage and activates the p19^Arf-p53 signaling pathway. (A) Western analysis of Ras, Myc, and p53 levels in Dicer^c/c MEFs either mock infected (−) or Ad-Cre infected (+) at 6, 11, and 15 dpi. Elevated p53 is observed in the Dicer-ablated cells, whereas Myc and Ras levels appear unchanged. Tubulin was used as a loading control for the Ras and p53 blot, as well as for the separate Myc blot. (B) DAPI and H2A.X staining of Dicer-ablated MEFs or control MEFs at 12 dpi. (C) The percentage of H2A.X-positive cells is increased in Dicer-ablated MEFs over time. Bar, 20 μM. (D) Western analysis of p21, p19Arf, and phosphorylated p53-Ser18 levels in Dicer^c/c MEFs mock infected (−) or Ad-Cre infected (+) at 6, 9, and 12 dpi. Elevated levels of p21 and p19Arf, and p53 activation in the Dicer-ablated cells. Tubulin is the loading control. (E) qPCR analysis of p53 target gene expression reveals that Dicer loss up-regulates p21, CyclinD2, PAI-01, and PAI-2. (F) Adriamycin induces a p53-mediated arrest in Dicer-ablated cells. (G) Morphology of Dicer^c/c MEFs at day 12 after Ad-control or Ad-Cre infection. Dicer^c/c MEFs either wt (left panels) or deleted for either In4a/Arf (center panels) or p53 (right panels). Deletion of Ink4a/Arf or p53 inhibited the premature senescence of Dicer-ablated MEFs. Bar, 400 μM.

Reduced primary cell proliferation and premature senescence in Dicer-ablated primary MEFs. (A) FACS indicates fewer Dicer-ablated cells undergo DNA replication. (B) Proliferation assay of Dicer-wt and Dicer-ablated cells reveals that Dicer loss inhibits cell proliferation. (C) Proliferation assay of Dicer-heterozygous and Dicer-ablated cells after induction of Cre activity. (D) SA-βGal staining of Dicer-wt or Dicer-ablated cells at day 12. Bar, 200 μM. (E) SA-βGal staining of Dicer^c/c MEFs after Ad-Cre or Ad-control infection. (F) DAPI-staining of DNA reveals dramatically altered heterochromatin in Dicer-ablated cells (right). Bar, 10 μM.

Deletion of Dicer induces cellular senescence in vivo. (A) SA-βgal staining of embryos after tamoxifen-induced Dicer ablation in utero reveals that loss of Dicer promotes senescence in E16 developing limbs. No senescence is seen in Dicer^c/wt embryos (right). (B) 3-mo-old sibling mice derived from K5-Cre x Dicer^c/c crosses. Dicer ablation results in fur loss after 12 wk. (C) Skin histopathology of mice retaining or ablated for Dicer by the K5-Cre transgene. Hematoxylin and eosin staining of skin sections followed by staining for SA-βGal activity. Dicer-ablated cells (bottom panels) are larger in size and display SA-βGal activity. Bar, 100 μM.