Animal Biotechnology and Modeling
231
usable (fertilized one- and two-cell ova).
However, fertilized ova were obtained with
enhanced efFciency compared to sponta-
neous ovulations in both young and aged
miniature pigs.
Breeding requirements for transgenic
pigs are critical, and successful manage-
ment calls for expertise in Felds ranging
from record keeping to animal husbandry.
With a gestation length of 114 days, and
an
interval
from
birth
to
puberty
of
6–12 months,
the
generational interval
seems extraordinarily
long,
particularly
when one is accustomed to the experimen-
tal progress observed in mouse studies.
±or xenotransplantation studies, the de-
sire to breed animals to homozygosity for
the transgene poses additional challenges.
Breeding schemes have been reported for
production efforts that, even under ideal
circumstances, lead to a three- to Fve-
year timetable (after founders have been
produced and identiFed), assuming no
lethality associated with transgene inte-
gration and insertional mutations. There
are
a
number
of
additional
questions
that may arise in the breeding efforts.
If transgenes interact, for example, their
effects may not be cumulative; indeed,
it might happen that such effects mask
one another, perhaps in competition for
trans-activators. Other issues involve sta-
bility of the genome as well as limiting the
gene pool in intricate breeding schemes.
Additionally, from a technical viewpoint,
evaluation of homozygosity of low copy
number or single-copy transgenic animals
can be very difFcult, with an additional
breeding/generational cycle needed to en-
sure the homozygous status of a transgene
in any particular animal.
In future experiments with domestic
animals, the ability to utilize embryonic
stem
cells
could
represent
a
tremen-
dous improvement over the microinjection
protocols currently in use. The ability to
‘‘knockout’’ or replace endogenous genes
has been an extremely valuable tool in
mouse genetics and modeling. Yet, char-
acterization and use of embryonic stem
cells for gene transfer has not worked to
date in any species other than the mouse.
The need for such technology in domestic
animal species is great, and the persever-
ance by the groups focused on this work
is exemplary.
4
Analysis of Transgene Integration and
Expression
4.1
Transgene Integration
When founder mice are three to four weeks
of age, biopsies are readily performed to
obtain representative tissues for analysis
of transgene integration and expression.
±or integration analysis in mice, typically
tail cuts (biopsies) are performed and the
tissue is digested with proteinase K fol-
lowed by DNA extraction using standard
protocols. Preliminary determination of
transgene integration by the PCR tech-
nique can be very useful when the target
sequence, the transgene, possesses unique
sequences (not endogenous to the genome
of the animal). However, given the extreme
sensitivity of PCR and the potential for
mispriming and false positives, other more
speciFc techniques should always be used
to conFrm the presence of the transgene
in PCR-positive samples. These include
DNA slot-blotting and Southern blot hy-
bridization, the latter being not only more
informative but also less likely to present
false positive signals. Additionally, the
transgene can be constructed to include
a molecular ‘‘tag,’’ a unique sequence
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