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. 2005 Feb 15;19(4):489–501. doi: 10.1101/gad.1248505

Figure 5.

Figure 5.

Reconstitution of Dicer expression and function in DCRΔ/Δ ES cells. (A) Western blot of extracts prepared from a homozygous (Δ/Δ) clone, a heterozygous (neo/+) ES cell clone, and two independent Dicer-reconstituted clones (DCR rec.). (Bottom) The Western blot was probed for Dicer protein using antisera (ab1414) and then stripped and reprobed for tubulin. (B) Northern analysis of miR-293 expression in total RNA from a control ES cell clone (neo/+), a Dcr-null clone (Δ/Δ), and Dicer-reconstituted clones performed as in Figure 1E. (Bottom) To demonstrate equal RNA loading, a segment of the EtBr-stained gel is shown. (C) Semiquantitative RT–PCR analyses of mesoderm- and ectoderm-specific differentiation markers. RNA was extracted and reverse-transcribed from EBs generated from DCRneo/+, DCRneo/neo, and two different DCRΔ/Δ clones at day 3, 5, and 8 of differentiation and analyzed for expression of differentiation markers bmp4, hnf4, gata1, and brachyury. Hprt transcripts were amplified as a loading control. (D) Semiquantitative RT–PCR of transcripts from centromeric minor satellite repeats in control (DCRneo/+ and DCRneo/neo), two mutants (DCRΔ/Δ), and Dicer-reconstituted (DCR rec.) ES cells. Digital photographs (negative image) of agarose gel analyses of the RT–PCR products for minor satellite and hprt transcripts are shown. Total RNA samples were treated with DNase I prior to reverse transcription. (E) DNA methylation analysis of genomic DNA derived from DCRneo/+, two DCRΔ/Δ, and two Dicer-reconstituted (DCR rec.) clones. Equivalent amounts of DNA were digested with HpaII and resolved in a 1% agarose gel. A Southern blot was performed with a probe specific for the minor satellite repeat (pMR150). The same blot was stripped and rehybridized with a mitochondrial DNA probe to demonstrate equal DNA loading. (F, right panels) Metaphase chromosomes from DCRΔ/Δ and Dicer-reconstituted cells were stained with a rabbit polyclonal antibody specific for trimethyl-H3K9. DAPI staining of chromosomes for each panel is shown on the left (100× magnification).