Biology Reference
In-Depth Information
(
Fig. 5.4
). 2i consists of basal medium supplemental with small molecule
inhibitors of MAPK signaling and of glycogen synthase kinase-3b and has
been demonstrated to maintain ES cells in a self-renewing ground state
(
Ying et al., 2008
). The 2i culture system, supplemented with LIF, has
enabled efficient culture and derivation of ES cells from all strains of mice
tested, including 129, C57BL/6, CBA, and NOD (
Batlle-Morera, Smith,
& Nichols, 2008; Kiyonari, Kaneko, Abe, & Aizawa, 2010; Nichols
et al., 2009; Ying et al., 2008
). Furthermore, reprogramming of mouse
somatic cells to induced pluripotency is also enhanced using 2i/LIF
(
Buehr et al., 2008; Li et al., 2008
). 2i/LIF has also enabled derivation,
for the first time, of germline-competent ES cells from rats (
Buehr et al.,
2008; Li et al., 2008
). We found that addition of 2i/LIF on the third day
of EG cell derivation led to a fivefold increase in EG cell derivation in
the mouse (
Leitch et al., 2010
). This same protocol allowed the derivation
of rat EG cells which self-renew in 2i/LIF conditions and exhibit properties
indistinguishable from rat ES cells, including extensive contribution to
chimeras (
Leitch et al., 2010
). Subsequently, rat EG cells have also been
established from later stage gonadal PGCs (
Northrup et al., 2011
). Intrigu-
ingly, these authors also reported that efficiency of EG cell derivation is
increased in rats carrying a mutation in
Dnd1
(
Northrup et al., 2011
).
Rat EG cells have also been reported to express Vasa (
Northrup et al.,
2011
), a notable difference to mouse EG cells which do not (
Toyooka
et al., 2000
). Crucially, rat EG cells derived from both early and gonadal
PGCs have been demonstrated to undergo germline transmission (
Blair
et al., 2012; Northrup et al., 2011
). This work establishes that the capacity
of PGCs to convert to pluripotent stem cells is not a mouse-specific phe-
nomenon and demonstrates that EG cells represent a feasible route to esta-
blishing transgenic technologies in other mammals. We propose that
application of 2i may prove a useful tool to extend these findings to non-
rodents, including humans. Although transgenic technologies could not
be applied to humans, establishment of na¨ve pluripotent human EG cells
would be useful in attempts to derive human PGC-like cells in culture
(
Hayashi et al., 2011
).
6. CONCLUSION
Our understanding of mammalian germ-cell development has
advanced significantly since the early observations of embryologists. Trans-
genic technologies have facilitated studies in the mouse, with relatively few
