220
Animal Biotechnology and Modeling
a substrate for the attachment of develop-
ing embryos, enabling them to develop
to a stage at which ES cells may be
harvested. Additionally, although not com-
pletely understood, culture conditions and
media/sera, as well as the handling of
ES cells before and during transfer proce-
dures, play critical roles in experimental
efFciencies.
±ollowing identiFcation of true ES cells
and subsequent gene-transfer procedures,
it is necessary to accurately and efFciently
identify
ES
cells
that
have
integrated
foreign DNA. In this regard, the
use
of marker-assisted selection schemes has
greatly simpliFed mouse experimentation.
Some
genes
were
easily
selectable
in
vitro
and therefore made screening less
difFcult, but most were not. In these
latter cases, PCR technology has been
used effectively to screen for homologous
recombination events. Mutated copies of
normal endogenous gene sequences have
been inserted in place of normal cellular
sequences,
and
PCR
techniques
were
used to screen for the novel mutants,
with the capability of detecting even a
single base-pair mutation. Hence, PCR
techniques have proven to be valuable tools
in selecting altered ES cell clones for use
in transgenic animal production.
ES cell–chimeric animals may indeed
harbor cells of both host embryo and ES
cell derivation, extending the study of germ
line transmission of desired transgenes
to one or more additional generations,
compared to most microinjection-derived
transgenic animals. Concerns associated
with the difFculty of line maintenance for
DNA
microinjection-derived
transgenic
animals are minimized, however, because
the ES cells have the potential to give rise
to multiple founder animals harboring the
same transgene at the same chromoso-
mal integration site. More recently, with
the addition of coculture techniques in-
volving tetraploid host embryos (eight-cell
stage to morula), founders can be de-
rived completely from the cocultured ES
cells. Hence, the founders are no longer
chimeras, having become ‘‘better engi-
neered’’ models.
Still, for most modeling employed to-
day, germ line transmission of transferred
genes in ES cell derived mice is influenced
to a great extent by the background ge-
netics of host and donor strains of mice.
Interestingly, germ line transmission is
routinely problematic in some strains but
not in others. The underlying biological
mechanisms responsible for such differ-
ences are not known.
While putative ES cell lines have been
identiFed
for
species
other
than
the
mouse,
production
of
germ
line
ES
cell derived/chimeric animals has proved
difFcult. If ES cells are identiFed that
could give rise to germline–competent
animals, it is probable that gene transfer
into such cells would be successful – based
on the wealth of available gene-transfer
literature. Thus, the most obvious question
to pose today is: Why has it been difFcult
to procure and utilize embryonic-derived
pluripotential
stem
cells
(ES
cells)
in
mammalian species other than the mouse?
Is it that present-day difFculties reflect
an evolutionary mutation in the mouse
genome, but not in other species, allowing
immortalization of pluripotent cells? Or
perhaps, might the idealized conditions
just be beyond our immediate grasp?
2.3.4
Nuclear Transfer (Cloning) Methods
Nuclear transfer, or cloning, refers to the
transfer of a nucleus from either an un-
differentiated stem cell or a differentiated
adult (somatic) cell to an enucleated oocyte,
thereby producing a now-viable embryo.
When this embryo is transferred into a
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