Animal Biotechnology and Modeling
221
suitable female, it can develop to term and
produce a live animal that is theoretically
genetically identical to that from which the
donor nucleus was obtained. If cloning
can be done with differentiated cells from
adult animals with desirable genotypic and
phenotypic superiority, then many genet-
ically identical copies can be produced as
an important experimental or commer-
cial resource.
Th
eF
r
s
tc
l
on
edm
i
c
ew
e
r
eid
en
t
iF
ed
in 1998 by the ‘‘Honolulu Method’’ of
nuclear transfer using adult cells (cumulus
cell nuclei). ±urthermore, serial nuclear
transfer resulted in cloned offspring from
previously cloned animals, demonstrating
conclusively that cells from cloned animals
were
not
different
from
normal
mice
in
their
ability
to
create
new
clonal
generations.
Embryonic stem cells, cumulus cells,
cultured
follicular
epithelial
cells,
tail
tip cells, Fbroblast cells, neuronal cells,
lymphocytes, fetal gonadal cells, Sertoli
cells, as well as blastomeres up to the
compacted morula stage have now been
successfully
used
in
nuclear
transfer
protocols. Yet surprisingly, at this time,
cloning by nuclear transfer using adult
donor
cells
has
not
been
successful
in
nonhuman
primates,
suggestive
of
a comparative species-speciFc biological
phenomenon; however, nuclear transfer
was pioneered in domestic animals, and
has now been demonstrated in a number
of species to date.
In the Honolulu method, metaphase II
oocytes are collected from the oviducts of
superovulated donors and enucleated us-
ing a holding pipette to stabilize the oocyte
and an enucleation pipette to aspirate the
chromosome–spindle complex. A nucleus
from a cumulus cell, with most of the cyto-
plasmic material removed, is then injected
into the oocyte. Electrofusion has also been
used in place of nuclear microinjection to
reconstitute the embryo. ±ollowing acti-
vation, the embryos are cultured to the
two-cell stage overnight and then trans-
ferred into oviducts of foster mothers.
The advantages of nuclear transfer over
other techniques include the following:
Most of the oocyte and embryo-handling
procedures have already been worked
out in the course of transgenic ani-
mal production.
Because cumulus cells are relatively
easily obtained and greater than 90%
are in G
0
/G
1
phase of the cell cycle,
(no
in vitro
culturing required), cumulus
cells are good nuclear donors for cloning
in mice.
Cloning of mice from ES cell nuclei
combines
the
possibility
of
genetic
manipulation of ES cells with cloning
by nuclear transfer.
Yet, there are still a number of limi-
tations to the current technologies. The
principal concern, reported by a number
of leading laboratories, is that cloning by
nuclear transfer remains relatively inefF-
cient. Well under 5% of ova and nuclear
transfers with cumulus or somatic donor
cells develop to term. Beyond the rel-
ative experimental efFciencies, potential
reprogramming and developmental conse-
quences of the various nuclear transfer re-
lated procedures are poorly characterized
to date. Similarly, influences of epigenetic
and mitochondrially encoded modiFers
will undoubtedly influence potentially un-
desirable outcomes.
2.3.5
Other Methodologies
Today, a host of techniques have been ap-
plied to the genetic modiFcation of mice.
In the late 1960s and 1970s, blastomere or
embryo aggregation and teratocarcinoma
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