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Fig. 1. Induction and monitoring of Gsc expression. (A) Our strategy for inserting
GFP gene into Gsc allele. Southern blots demonstrate correct
insertion of Gsc-gfp transgene and removal of Neo cassette. (B-F) Gsc
induction under various culture conditions. Cultures were basically performed
on collagen IV-coated dishes, except embryoid body (EB) formation experiments
(right panels in B, part i and F). Gscgfp/+ES cells were
cultured for 4 days (D4) or 6 days (D6). (B, part i) Day 4 FACS analysis of
cells cultured in serum containing medium (SCM). (B, part ii) RT-PCR
expression analysis of GFP+ and GFP- population induced
in SCM for 4 days. The specific molecules for the organizer were only
expressed in GFP+ population. (B, part iii) GFP expression in
tetraploid embryo. GFP expression was strongly detected in the organizer
region of mid-streak stage embryo. (C-F) SF-O3 serum-free media (SFM) were
used in Gsc induction. (C, part i) Selective induction of Gsc-GFP.
Ninety-three percent of cells expressed Gsc-GFP on day 6 in the monolayer
culture with activin. (C, part ii) RT-PCR analysis of GFP+ and
GFP- population on day 4. (C, part iii) Expression pattern of
visceral endoderm markers. Upper panel shows RT-PCR analysis of total cells
differentiated in SFM with activin for 4 days. Lower panel shows that of EBs
differentiated in SCM for 4 days. (C, part iv) Immunostaining showing that
almost all of cells on day 6 in SFM plus activin expressed brachyury protein.
(D) Effect of Nodal on the generation of Gsc+ cells. Nodal showed a
similar activity to induce Gsc+ cells. (E,F) Bmp4 and serum
inhibited the generation of Gsc+ cells even in the presence of a
large amount of activin. The culture with EB formation is less efficient for
the generation of Gsc+ cells even in SFM (F), than the monolayer
culture (C). The units displayed for activin (Act), nodal and Bmp4 are
ng/ml.
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