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Fig. 6. Smad5 knockdown in mouse EBs results in reduced Eklf and Gata1
expression. (A) Schematic of the GFP-Intron-miR transgene after
site-specific, uni-directional integration into the Ainv18 homing site,
including the location of the miR-30 backbone and shRNA insertions into the
GFP intron. (B) Flow cytometric analysis of GFP expression in the Smad5
shRNA-1 Ainv18 ES cell clone in doxycycline treated (24 hours, +dox) or
untreated (-dox) EBs harvested at day 5 of differentiation. (C)
Analysis of Smad5, Eklf and Gata1 expression by qRT-PCR in
the Smad5 shRNA-1 clone. Untreated (-dox) or doxycycline-treated (24 hours;
+dox) cells were harvested at day 5 of EB differentiation. To enrich for
transgene-expressing populations, cells from untreated EBs were FACS sorted
for GFP-negative cells (-dox/GFP-), whereas cells from dox-treated
EBs were FACS sorted for GFP expression (+dox/GFP+) prior to being
monitored for RNA expression. Expression levels were normalized to
Gapdh and untreated samples were set to 1. (D) Analysis of
Smad1, Smad5, Eklf, Gata1 and Gata2 expression by qRT-PCR in
the shRNA-1 clone in cells harvested from EBs at day 5 of differentiation
without FACS sorting. RNA was isolated from untreated (-dox) or
doxycycline-treated (24 hours, +dox) cells as indicated. Expression levels
were normalized to Gapdh and each untreated sample was set to 1.
Arithmetic mean ±s.d. (E) Western blot analysis of Gata2, Eklf,
Gata1 and GFP protein expression in shRNA-1, shRNA-2 and shRNA-control Ainv18
ES cell clones harvested at day 6 of EB differentiation. Lysates were prepared
from unsorted EBs that had been treated with (+dox) or without (-dox)
doxycycline for 48 hours. Hsp90 expression levels were used as a loading
control. Molecular mass (kDa) markers for each blot are shown to the left.
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