Figure 2.
Dicer-deficient ES cells retain ES cell characteristics but fail to differentiate. (A) Phase-contrast micrographs of heterozygous (neo/+) and homozygous (Δ/Δ)ES cell colonies (indicated by arrows) grown on a feeder layer of MEFs. (B, top panel) RT–PCR analysis of the ES cell-specific α6-integrin isoform (306 bp). (Bottom panel) Quantitative RT–PCR analysis of oct-4 transcripts normalized to hprt transcripts. The averages and standard deviations of three experiments are depicted in the graph. (C) Size of teratomas following injection of the indicated ES cells into nude mice is plotted as a function of time (weeks post-implantation). The averages from five injected mice are shown. (D) Light micrographs of paraffin sections from day 12 EBs stained with hematoxylin-eosin. EBs derived from heterozygous (DCRneo/+), homozygous (DCRneo/neo), and Dicer-deficient ES cells (DCRΔ/Δ) are shown in the top panel (10×). In the lower panel, higher magnification images (40×) of a portion of the same EBs are displayed. (E) 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. (F) Quantitative real-time PCR analysis of oct-4 transcripts normalized to hprt transcripts in EBs at days 3, 5, 8, 10, and12 of differentiation. The averages and standard deviations of three experiments are depicted in the graph.