|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
Fig. 4. Gata factor and Smad binding motifs regulate Eklf expression in
vivo. (A) Schematic of the Peklf-GFP reporter construct and its
four derivatives, which were targeted to the Ainv18 ES cell line, that carry
two GATA motif mutations (2xG/A) in the upstream enhancer (Peklf-2xG/A-GFP),
or one GATA motif mutation in the proximal promoter (Peklf-G/A-GFP), two Smad
binding motif deletions (2x
SBM) in the upstream enhancer
(Peklf-2xSBM-GFP), or an insertion of Eklf intron 1
(Peklf-intron-GFP). Positions of Eklf cis-regulatory elements and
translation start codons (ATG) are indicated and color-coded as in
Fig. 3. (B-E) qRT-PCR
analysis of GFP transgene expression in the four derivative Ainv18 ES cell
reporter clones between days 4 and 7 or 8 of EB differentiation. Expression
levels were normalized to 18S rRNA or Gapdh. For Eklf and
GFP, normalized levels are presented relative to a value of 1 on EB
day 4, whereas for Gata2 (G2) and globin βH1 (bh1) the maximum
expression level per gene is set to 1. Arithmetic mean ±s.d.;
*, 0.01<P<0.05, n=3; **,
0.001<P<0.01, n=3. (Left) Endogenous Eklf
(red) and GFP (green) expression from the Peklf-GFP clone,
differentiated in parallel in each experiment, is shown for cross-comparison.
(Right) Endogenous Eklf (red) and GFP (green) expression
from each mutant clone (Peklf-2xG/A-GFP, Peklf-G/A-GFP,
Peklf-2xSBM-GFP, Peklf-intron-GFP) is shown along with that of
Gata2 (black) and globin βH1 (orange). Within each panel, high
levels in Gata2 expression are indicative of a progenitor population in
differentiating EBs prior to day 5.5, after which the onset of globin βH1
expression is indicative of erythroid commitment (expression patterns as in
Fig. 2A).
|
