224
Animal Biotechnology and Modeling
gene expression – in deference to the en-
suing cascade of endocrine events that
unfolded. In contrast, efforts at using do-
mestic farm animals as bioreactors or in
transplantation-related efforts have contin-
ued to progress, and viable products are
expected to petition for regulatory approval
shortly. Whether they will gain market ac-
ceptance is still largely unknown.
3.2
Methodology
Today,
DNA
microinjection,
retroviral
transfection, nuclear transfer and passive
transfer procedures (e.g. sperm-mediated
transfer) have been used to successfully
produce
transgenic
livestock.
Genetic-
engineering capabilities will continue to
mature, in concert with advances in whole-
animal and somatic-cell techniques (in-
cluding liposome-mediated gene transfer,
jet injection, and particle bombardment).
Envisioned progress and expansion of our
existing knowledge base will allow us to
better maximize and engineer production
traits in farm animals in a most effec-
tive fashion.
Along the same lines, procedures to aug-
ment gene-transfer efFciencies have a high
priority, especially where long gestational
intervals prove problematic. Protocols have
been developed to permit removal and
screening of individual blastomeres from
preimplantation embryos maintained in
culture prior to transfer to recipient fe-
males. Sensitive methods involving the use
of conventional and real-time PCR allow
analysis of DNA puriFed from individual
blastomeres and subsequent identiFcation
of those embryos that bear genetic modi-
Fcations of interest. Use of such methods
offers the potential to greatly increase the
efFciency associated with production and
selection of transgenic farm animals and
to thereby signiFcantly reduce the associ-
ated costs.
3.2.1
DNA Microinjection
DNA microinjection was the Frst method
used to successfully produce transgenic
livestock. Although involved and at times
quite tedious, the steps in the development
of transgenic models are relatively straight-
forward. Once a speciFc gene sequence is
cloned and characterized, sufFcient quan-
tities are isolated, puriFed, and tested in
cell culture. If
in vitro
mRNA expression of
the gene is identiFed, the appropriate frag-
ment is linearized, puriFed, and readied
for preliminary mammalian gene-transfer
experiments. The DNA microinjection ex-
periments
are
Frst
performed
in
the
mouse model. While the transgenic mouse
model will not identify likely phenotypic
expression patterns in domestic animals,
we have not observed a single construct
that would function in a pig when there
was no evidence of transgene expression
in mice. Therefore, preliminary
in vitro
and/or
in vivo
experimentation has been
a crucial component of any gene-transfer
experiment in domestic animals.
±ollowing
conFrmation
of
transgene
expression in mice, it then becomes cost-
e
f
f
e
c
t
i
v
et
oin
i
t
i
a
t
eDNAm
i
c
r
o
in
j
e
c
t
i
on
experiments in other species. In mouse
experiments, less than two months is
required
from
the
time
the
puriFed
construct
is
ready
for
microinjection
through weaning of founder pups. In
contrast, for pig experiments, one month
to a year is required for a sufFcient number
of DNA injections and recipient transfers
to
ensure
the
likelihood
of
success.
Experimental efFciencies coupled with a
long
generational interval (i.e.
114 day
gestation period, 21–28 day lactation, and
onset
of
puberty
between 6–9 months
of age), reflect the efforts necessary to
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