Animal Biotechnology and Modeling
219
considerations, and potential for unde-
sired genetic recombination that may alter
the replicative characteristics of the retro-
virus, (3) low copy number integration,
(4) a high frequency of mosaicism (which
necessitates subsequent outbreeding of
founder animals to generate pure lines),
and (5) possible interference by retroviral
sequences on transgene expression.
2.3.3
Embryonic Stem Cell Technology
Gene transfer has been used to produce
both random and targeted insertion or ab-
lation of discrete DNA fragments into the
mouse genome. For targeted insertions,
where the integration of foreign genes is
based on a recombinational gene inser-
tion with a speci±c homology to cellular
sequences (termed
homologous recombina-
tion
), the ef±ciency of DNA microinjection
is extremely low. In contrast, the use of ES
cell transfer into mouse embryos has been
quite effective in allowing an investigator
to preselect a speci±c genetic modi±ca-
tion, via homologous recombination, at a
precise chromosomal position. This pre-
selection has led to the production of mice
that (1) incorporate a novel foreign gene
into their genome, (2) carry a modi±ed
endogenous gene, or (3) lack a speci±c en-
dogenous gene following gene deletion or
‘‘knockout’’ procedures.
Technologies involving ES cells, and
more
recently
primordial
germ
cells,
have been used to produce a host of
mouse
models.
Pluripotential
ES
cells
are derived from early preimplantation
embryos and maintained in culture for
a suf±cient period for one to perform
various
in vitro
manipulations. The cells
may be injected directly into the blastocoel
of
a
host
blastocyst
or
incubated
in
association with a zona-free morula. The
host embryos are then transferred into
intermediate hosts or surrogate females
for continued development. Currently, the
ef±ciency of chimeric mouse production
results in about 30% of the live-born
animals containing tissue derived from
the injected stem cells. This ability to
produce
‘‘chimeric’’
animals
using
ES
cells has given researchers another tool in
their armamentarium for the production
of
transgenic
animals.
In
this
set
of
techniques, the power of gene-transfer
technology has been catapulted forward
because such processes allow for targeted
insertions into the genome. Such targeting
is
extremely
important,
particularly
in
areas
of
gene
therapy
and
correction,
wherein
previous
technologies
allowed
only for random integration events.
ThegenomeofESce
l
lscanbemanipu-
lated
in vitro
by introducing foreign genes
or foreign DNA sequences by techniques
including electroporation, microinjection,
precipitation
reactions,
transfection,
or
retroviral insertion. The use of ES cells to
produce transgenic mice faced a number
of procedural obstacles before it became
competitive with DNA microinjection as a
standard technique in mouse modeling.
The ±rst procedural obstacle in creating
ES cell–derived transgenic animals was
the extreme dif±culty associated with the
production and maintenance of ES cell
lines. This problem lies in the general in-
ability of embryos of most mammalian
species, other than mice, to survive
in
vitro
. A standard technique for producing
murine ES cells involves culture of em-
bryos
in vitro
, up to and beyond the point
at which they would normally implant in
the uterus. In mouse-embryo culture, this
period is mimicked through coculture on
±broblast monolayers or by the inclusion
of various growth factors, (leukemia in-
hibitory factor [LIF], ciliary neurotropic
factor [CNTF], etc.) in the medium. In
the case of coculture, ±broblasts serve as
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