Animal Biotechnology and Modeling
215
for new investigators. The overall efF-
ciency of many procedures will vary, as
will the cost–beneFt ratios, however, one
must not let the formidable challenges of
gene-transfer experimentation outweigh
the primary reason for instituting such
studies – the desire to develop a biological
model with speciFc genetic characteristics
in a whole-animal environment.
2
Production of Transgenic Laboratory
Animals
2.1
Choice of Animals
Therelativeimportanceofusingparticular
s
t
r
a
i
n
so
rb
r
e
e
d
so
fa
n
im
a
l
si
ng
e
n
e
-
transfer experimentation will vary dra-
matically according to the species under
consideration. Probably the most complex
system is encountered in the production of
transgenic mice, simply because so much
work has been done with murine species.
Here, well-documented differences in re-
productive productivity, behavior, related
husbandry requirements, and responses
to various experimental procedures play
major roles in determining the efFciency
and the degree of effort associated with
production of transgenic founder animals.
A general discussion of these factors there-
fore serves as an appropriate starting point
for understanding the many processes and
procedures that must be evaluated and
monitored when considering production
of transgenic animals.
DNA microinjection, the most direct
and reproducible method for producing
transgenic animals, necessitates that sev-
eral ‘‘pools’’ of animals be maintained for
speciFc purposes. Most commonly, donor
females are induced to superovulate by
means of a regimen of injections of preg-
nant mare serum gonadotropin (PMSG,
to stimulate follicular growth and devel-
opment) followed by human chorionic
gonadotropin (hCG, to induce ovulation
of mature eggs). These donor females are
then mated to fertile males, and large num-
bers of fertilized one-cell eggs (‘‘zygotes’’)
are obtained surgically. Alternatively, ova
may be collected from donor females and
then subjected to
in vitro
fertilization (IV±)
to obtain zygotes. In either case, the DNA
construct, in a buffer solution, is mi-
croinjected into the male pronuclei of
the fertilized eggs. Ova that survive mi-
croinjection are surgically transferred to
the reproductive tracts of hormonally syn-
chronous female recipients, which carry
the embryos to term. In mice, DNA from
offspring is isolated from tissue samples
(e.g. tail biopsies) obtained at weaning and
analyzed by Southern blot analysis and/or
methods based on the polymerase chain
reaction (PCR) to determine which indi-
viduals carry the transgene.
Certainly, the relative costs of different
strains of mice that are chosen to serve
as
gamete
donors
or
recipients
is
a
factor. This must be balanced with the
background genetics of the various strains
and
the
effects
these
may
potentially
exert
on
expression
of
the
integrated,
foreign DNA construct (the transgene) and
the resulting phenotype. Strains used for
donor females are chosen for (1) numbers
of eggs ovulated in response to hormonal
superovulation regimen, (2) the relative
uniformity of this response within the
strain, (3) the viability of superovulated
eggs both before and after microinjection,
(4) the ease with which fertilized eggs
are visualized and recovered from the
reproductive tract, and (5) the morphology
and/or size of pronuclei (which affects the
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