|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
Fig. 4. Identification of Hex targets in ES cells. (A) Schematic
representation of the ES cell lines used in the Hex target screen. (a) cDNAs
inserted into the Rosa26 locus. (b) Strategy for the generation of clonally
related cell lines. Top line illustrates the two Rosa26 targeting constructs
used to generate parental control cell lines that do not express the transgene
because of the triple PolyA stop cassette. Transfection of these lines with
CRE-recombinase induces recombination between the two LoxP sites, removing the
stop cassette and allowing expression of the indicated cDNA. This strategy
ensures that all the transgenic lines are derived from a clonally related
control. SA refers to Rosa26 splice acceptor. (B) Outline of the Hex
target screen. RRosa is recombined Rosa. (C) Profile of gene expression
across a series of treatment comparisons. RHETVPcl1+/-4-OHT refers to the
average ratio of gene expression levels in Rosa HexERT2-
VP2 in the
presence and absence of 4-OHT for clone 1. RRHETVPcl1+/-4-OHT refers to the
average ratio of gene expression levels in the recombined Rosa
HexERT2-VP2 in the presence or absence 4-OHT for clone 1.
RHETVPcl2+/-4-OHT refers to the average ratio of gene expression levels in
Rosa HexERT2-VP2 in the presence or absence of 4-OHT for clone 2.
RRHETVPcl2+/-4-OHT refers to the average ratio of gene expression levels in
the recombined Rosa HexERT2-VP2 in the presence or absence of 4-OHT
for clone 2. RH+/-CRE refers to the average ratio of gene expression levels in
Rosa Hex in the presence or absence of CRE. The five candidate genes obtained
are shown in black against the background of all gene expression profiles. The
gene expression averages in this plot are based on samples considered to
represent undifferentiated cultures as judged by expression of key markers
such as Oct4. (D) List of candidate Hex target genes. (E) Tle4
and Nodal expression is induced by the addition of 4-OHT to the cultured
HexERT2-VP2-expressing cells. Cells were cultured and treated with 600
nM 4-OHT in serum-free media and expression of the genes analysed by real-time
RT-PCR. Removal of serum from ES cell cultures appears to remove a suppressor
of HexERT2-VP2 activity and consequently we observe more robust target
gene induction. Values are normalised to the expression level of ß-actin
and presented as relative copy number.
|
