226
Animal Biotechnology and Modeling
development in species beyond the mouse
model. This is because a technological
barrier was surpassed that allowed for
speciFc
in vitro
manipulations that lead
to targeted genetic modiFcations in Frst-
generation founder animals. Prior to the
mid-1990s, it was not possible to pro-
duce germ line–competent transgenics in
mammalian species other than in mice,
using any technique other than DNA mi-
croinjection (that only allowed for random
and imprecise integration of transgenes in
founder animals). Unfortunately, relative
efFciencies for nuclear transfer experi-
m
e
n
t
a
t
i
o
ns
t
i
l
lp
a
l
e
si
nc
om
p
a
r
i
s
o
nt
o
conventional DNA microinjection. How-
ever,
while
nuclear
transfer
might
be
considered inefFcient in its current form,
major strides in enhancing experimen-
tal protocols within the next few years
are envisioned, comparable perhaps to
the early advances in DNA microinjection
technology.
Nuclear transfer involves a technique in
which nuclei of various cell origins are in-
troduced as noted in Sect. 2 above (±ig. 2).
The successful ‘‘cloning’’ of a sheep was
reported in the mid-1990s and rekindled
the imagination of researchers struggling
with microinjection and related technolo-
gies. Yet, while nuclear transfer might be
considered inefFcient in its current form,
major strides in enhancing experimental
protocols have been envisioned, and with
recent
in vitro
maturation of pluripotential
cell lineages, the marriage of a number of
existing techniques may prove fruitful in
the near term.
The techniques for nuclear transfer in
domestic animals mirror what
is cur-
rently employed for mouse studies. Nuclei
from quiescent (G
0
) cells (or the intact
Blastocyst
Oocyte
Removal of
chromosomes
and polar bodies
Electrofusion
of nucleus and
oocyte
Inner cell mass
Nuclear donor
cells cultured
Nuclear
transfer
Fig. 2
Cloning sheep by nuclear
transfer. In this example, inner cell mass
cells from blastocyst stage embryos (or
cells from somatic tissues) are obtained
and propagated in culture. If so desired,
the culture capability here, allows one to
genetically modify cells prior to transfer.
Selected cells are then used as nuclear
donors for transfer into enucleated
oocytes. In contrast to DNA
microinjection, a fusion step is generally
employed to fuse the transferred nuclei
and enucleated oocytes. Here,
electrofusion is used to fuse couplets (a
donated nucleus plus enucleated
oocyte) that are then transferred to
recipients for the remainder of
gestation. (Reprinted from Pinkert, C.A.,
(2000)
Reproduction in Farm Animal
s,
7th ed., Hafez, E.S.E. and Hafez, B.,
Eds., Williams & Wilkins, Baltimore,
Chap. 21, pp. 318–330. With
permission.)
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