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Fig. 3. Derivation of PouV clonal cell lines. (A) Experimental
strategy to expand and analyse PouV clonal cell lines. (B) Cell lines
stained for AP activity (red). The –Oct4/+LIF cells are at passage 10
(p10), apart from the ZHBTc4 line, shown 72 hours after Oct4 transgene
repression. The –Oct4/-LIF cells are at p10, 5 days after LIF
withdrawal. The +Oct4/+LIF cells are at p10, 5 days after Oct4 transgene
re-expression. Scale bars: 100 µm. (C) Real-time RT-PCR analysis for
PouV expression. RNA was collected at p10. Values represent the mean
expression levels calculated from all available cell lines: Xlpou25
(n=2), Xlpou60 (n=8), Xlpou91 (n=10), mouse Oct4
(n=10). (D) Real-time RT-PCR analysis of ES cell markers genes
from PouV ES cell lines. RNA was obtained at p10. Values represent the mean
expression levels calculated from at least two independent cell lines.
(E) Real-time RT-PCR analysis of ZHBTc4 ES cells following the
shut-down of Oct4 transgene expression. RNA was collected at the indicated
timepoints following shut down of Oct4 transgene expression. Values were
normalized to ß-actin and the relative change in gene expression for the
marker genes analysed was calculated by dividing the –Oct4 values by the
+Oct4 control value. Experiments were carried out in triplicate. (F)
Real-time RT-PCR analysis of PouV protein overexpressing ES cells. RNA was
collected from cell lines maintained in the absence of Oct4 and from cell
lines 72 hours after Oct4 transgene re-expression. Values were normalized to
ß-actin and the relative change in gene expression of the marker genes
analysed was calculated by dividing the +Oct4 values by the –Oct4
values. Experiments were carried out in triplicate.
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