Animal Biotechnology and Modeling
229
of DNA into the cytoplasm of one- and
two-cell ova. Fortunately, it was found that
centrifugation of pig ova resulted in strati-
±cation of the cytoplasm, rendering pronu-
clei and nuclei visible under differential
interference contrast or Nomarski/Smith
microscopy. Even though the situation was
greatly improved, centrifugation failed to
reveal the nuclear structures in 15–33% of
fertilized eggs. However, when using con-
ditions initially optimized for transgenic
mouse production, DNA microinjection
into pronuclei/nuclei resulted in nearly a
10% yield of transgenic offspring (vs. total
live-born/viable offspring).
The proportion of transgenic swine that
develop from microinjected ova, which
varies from zero to about 12%, is generally
much lower than that observed in mice.
The survival of microinjected pig embryos
is related to several factors including the
developmental stage of ova injected, the
duration of
in vitro
culture, synchrony of
ova donors and recipients, the number
of ova transferred, and donor age. Other
factors that have been shown to influence
the developmental ef±ciency of transgenic
mouse production – including technician
pro±ciency and embryo handling/transfer,
pipette dimensions, DNA preparation, and
the viability of microinjected ova – readily
influence transgenic production ef±ciency
forallotherspecies.
If
a
foreign
gene
fragment
is
inte-
grated into a domestic animal’s genome,
the gene copy number and orientation
(head-to-head vs. head-to-tail arrays) vary
considerably, as might be anticipated from
the mouse studies. In addition, mosaic
animals are sometimes produced, making
analysis of transgene integration more dif-
±cult. Recently, the results of PCR analyses
of founder animals were useful in detect-
ing extreme mosaicism, where a given
transgene is found in only a proportion
of the animal cells. While the identi±ca-
tion of transgenic founders (both mosaic
and nonmosaic) can be enhanced using
PCR analyses, con±rmatory DNA slot-blot
or Southern blot analyses are necessary
to avoid extreme and costly breeding ef-
forts to identify germ line transmission of
the transgene.
In one set of experiments, approximately
70% of pigs harboring a growth hormone
gene construct (as determined by Southern
blot analysis) expressed the transgene-
encoded growth hormone. Failure of the
remaining 30% of transgenic swine to
express the foreign gene was attributed
to integration of the transgene into an
inactive chromosomal locus or alteration
of
the
transgene
sequence
during
its
integration. Of the pigs that did express the
transgene, the level of expression appeared
to vary greatly among individuals. While
cis- and trans-acting factors are obviously
important influences affecting transgene
expression, the
integration
site
of
the
transgene is equally critical. Transgene
transcription rates are likely influenced by
the level of activity present at the locus, or
the site of integration, and the properties
of enhancer sequences located in genes
flanking the transgene.
While it is not unreasonable for an inves-
tigator to suggest that 40 transgenic mice
can be produced in a week’s worth of DNA
microinjections, there are signi±cant dif-
ferences in undertaking pig experiments.
In the pioneering collaboration of the Uni-
versity of Pennsylvania, the University of
Washington, and the United States Depart-
ment of Agriculture, which ±rst reported
on the production of transgenic pigs, more
than four years went into amassing the
experimental results that culminated in
the production of 40 founder transgenic
pigs. The 55% of founder animals that
expressed transgene-encoded mRNA was
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