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Fig. 2. EG cell production from AKT-MER-expressing PGCs under standard
conditions. (A) Procedure for EG cell derivation. Cell suspensions
of E11.5 gonads were seeded onto Sl/Sl4-m220 feeder cells that
express the membrane-bound form of SCF, and cultured with LIF and bFGF
(primary culture). 4OHT and bFGF were added only during primary culture. At
day 5 of primary culture, whole wells were trypsinized and passaged onto fresh
Sl/Sl4-m220 layers. Secondary culture was carried out with LIF for
5 days. The whole wells were finally passaged onto MEFs and cultured with LIF
(tertiary culture). At day 5 of tertiary culture, the wells containing EG
cells were counted to determine the derivation efficiency. The EG cells at
tertiary culture were propagated to stable cell lines on MEFs in the presence
of LIF. A fraction of EG cells in the primary cultures (primary EG cells) gave
rise to stable EG cell lines by this whole-well passage procedure. The
efficiency is detailed in the legend of
Table 2A. (B) Alkaline
phosphatase staining of primary EG cell colonies derived from wild-type PGCs
cultured without 4OHT (left) and of transgenic PGCs cultured with 100 nM 4OHT
(right). The EG cell colonies were multilayered and positive for alkaline
phosphatase activity. Scale bar, 100 µm. (C) Increased number of
AKT-MER-expressing PGCs. The cells of 0.02 embryos were added to each well,
and 30-40 PGCs attached to feeder cells. The percentage change in the PGC
population was calculated by dividing the number of PGCs at day 3 by the
number of attached PGCs. The 4OHT-treated transgenic PGCs proliferated
significantly more than untreated transgenic PGCs (mean±s.e.m.;
*P<0.005, #P<0.0005, Student's
t-test; n=6) and wild-type PGCs cultured with the
corresponding concentrations of 4OHT (P<0.005,
P<0.0005 and P<0.005 for 100 nM, 300 nM and 1000 nM,
respectively). (D) Increased primary EG cell colony formation by E11.5
AKT-MER-expressing PGCs. A primary EG cell colony was defined as a
multilayered, alkaline phosphatase-positive colony of more than 20 cells, as
described previously (Kimura et al.,
2003; Moe-Behrens et al.,
2003). Colony formation was significantly higher for 4OHT-treated
transgenic PGCs than for untreated transgenic PGCs (mean±s.e.m.;
*P<0.005, #P<0.0005, Student's
t-test; n=6) and wild-type PGCs cultured with the
corresponding concentrations of 4OHT (P<0.0005,
P<0.005 and P<0.0005 for 100 nM, 300 nM and 1000 nM,
respectively).
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