216
Animal Biotechnology and Modeling
ease and speed with which eggs can be
microinjected).
The strain chosen for embryo transfer
recipient use is evaluated for (1) parenting
ability, (2) docile behavior (which allows
for inspection of pups or cross-fostering
after birth), (3) ease of embryo transfer
procedures, and (4) the regularity with
which females exhibit readily detectable
vaginal plugs and undergo a pseudopreg-
nant response when mated with vasec-
tomized males.
Obviously, the genetic background of the
strain used for vasectomized males (and
also recipient females) is less important,
since these animals do not contribute to
the genetic composition of the transgenic
offspring. Strains are favorably considered
in which vasectomized males exhibit libido
and are able to form visible copulatory
plugs in the recipient females both fre-
quently (two to three matings/week) and
with regularity over a period of about
12 months.
Conversely, intact males are chosen for
their (1) libido, (2) absence of nonmating
aggressiveness when placed with families,
(3) fertility and capacity to produce visi-
ble copulatory plugs, and, if serving as
IVF donors, (4) sperm concentration and
quality. Finally, it is desirable to maintain
a pool of synchronous, pregnant females
that can serve as foster mothers in the
event that recipient females begin to can-
nibalize pups or fail to adequately nurse
their offspring.
For most other nonmurine species, it
will remain important to use donor fe-
males that respond well to hormonal syn-
chronization and superovulation, and to
have recipients that are able to carry fetuses
to term and care for neonates appropri-
ately. However, differences in methods
used for hormonal synchronization, selec-
tion of proestrus females, and evaluation of
other forms of reproductive behavior may
differ signi┬▒cantly from those identi┬▒ed
for mice.
2.2
Gain-of-function, Loss-of-function and
Conditional Modeling
With
advances
in
the
characterization
of factors that control gene expression,
gene-transfer technology has become an
important means of dissecting gene regu-
lation and developmental pathways
in vivo
.
Normally, endogenous gene function is
regulated by a variety of molecular fac-
tors. Analysis of transgenic animals has
illustrated molecular underpinnings and
events associated with developmental tim-
ing, tissue distribution, and consequences
of modifying gene expression. Addition-
ally, transgenic animals have also proven
quite
useful
in validating an array
of
in vitro, in vivo
, and theoretical model
systems. Today, there are multiple mod-
eling capabilities that allow a researcher
to develop a given hypothesis revolving
around a gain-of-function experiment (e.g.
DNA microinjection, retroviral-mediated
transgenesis, or knockin gene targeting
procedures are used), a loss-of-function
model (e.g. knockout gene targeting or
overexpression of cellular toxins may be
envisioned) or conditional systems (e.g.
use of traditional temporally or spatially
regulated transgenes, or where Cre-lox
or
other
in vivo
recombinase
systems
are employed).
2.3
Methodologies
2.3.1
DNA Microinjection
The microinjection method, referred to
earlier, involves the use of micromanipula-
tors and an air- or oil-driven microinjection
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