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Fig. 3. Reduced proliferation of Sall4-null ES cells. (A)
Northern blot analysis showing the reduction of Sall4 upon
Sall4-siRNA treatment. KD (knockdown), treated with Sall4-siRNA; NC (negative
control), treated with control siRNA. (B) Transiently reduced
proliferation of ES cells upon Sall4-siRNA treatment. Cells were counted in
triplicate. (C) Conditional disruption of Sall4 in ES cells.
When a Sall4-IRES-Hyg vector was introduced into cells heterozygous
for a floxed allele of Sall4 (flox/+), both alleles were targeted
with a similar frequency, resulting in two types of cells: flox/- and +/-.
Upon infection with adenovirus expressing Cre, flox/-cells became almost
Sall4-null by day 3, determined by western blot (lowest panels).
White triangle, Frt; black triangle, loxP. (D) Reduced proliferation of
Sall4-null ES cells. Cell expansion rate over 16 days is shown, using
Sall4-null (-/-) versus flox/-cells obtained upon the same Cre
treatment. Analysis was carried out in triplicate. (E) Reduced S phase
and increased G1 phase in the Sall4-null ES cells. Consistent data
were obtained from two independent experiments using three Sall4-null
cells, and the representative data is shown. (F) Normal morphology and
positive staining of Oct3/4 of a Sall4-null ES colony. (G)
Northern blot analysis of Sall4-deficient ES cells. Two heterozygous
(flox/-), and two Sall4-null ES (-/-) clones are shown. (H)
Chimeric embryo formation from Sall4-null ES cells transfected with
GFP. (left) High contribution of GFP-expressing cells in the E7.5 embryo.
(Right) A section of the chimera was stained by an anti-GFP antibody and
detected by DAB.
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