232
Animal Biotechnology and Modeling
that is easily detected and has minimal
similarity to any endogenous sequence.
This strategy is especially useful when the
transgene itself is similar or nearly iden-
tical to an endogenous sequence. In such
cases, another strategy that may be help-
ful is the introduction of new restriction
sites into the transgene, without perturb-
ing function, so that restriction fragment
length polymorphism (RFLP) analysis can
be used to distinguish the transgene from
its endogenous counterpart. Finally, in
some instances phenotypic screening is
possible if the expression of the trans-
gene leads to an identi±able change in
the appearance of the animal. Phenotypic
screening by coinjection of a second trans-
gene that gives rise to a speci±c phenotype
can also be useful, since cointegration
is much more likely than integration of
the phenotypic marker gene alone. Later,
however, the integration of the transgene
of interest must be con±rmed by other
means.
Other concerns related to the analysis
of transgene integration include determi-
nations of copy number of the transgene
per cell, orientation of tandemly arranged
copies, and the presence of multiple in-
tegration sites. These questions can be
addressed by Southern blot hybridization
following digestion of the genomic DNA
with appropriate restriction enzymes. In
addition, the functionality of the transgene
is an important consideration in the analy-
sis of transgene integration. However, this
aspect is usually dealt with in the analy-
sis of transgene expression (detection of
a functional mRNA transcript or protein
product). Finally, the critical question of
whether the transgene is integrated into
thegermlineofthefounderanimalandis
heritable as a stable genetic element must
be answered.
4.2
Transgene-encoded mRNA Expression
The analysis of expression of the transgene
is,
of
course,
absolutely
essential
in
determining the utility of the transgenic
animals produced. As with integration
analysis, the presence or absence of similar
or identical endogenous counterparts will
determine, to a degree, the strategies that
may be most useful. For transgenes that
are unique (no endogenous counterpart)
or contain some unique sequences, the
strategies
that
can
be
used
are
more
straightforward. The presence of a novel
mRNA transcript or a unique protein (or
enzyme activity) is more easily determined
than in situations involving transcripts
or protein products that are very similar
to endogenous transcripts
or proteins.
As with integration analysis, molecular
‘‘tags’’
are
also
sometimes
useful
in
that the
transcripts will contain some
unique
identifying
sequence
that
can
be readily and unequivocally determined.
Once
again,
care
must
be
taken
to
ensure that any additions or deletions
designed to aid in analysis do not interfere
with expression.
Technically, the most critical step in
analyzing
transgene
expression
is
the
isolation of RNA. Great care must be
taken to avoid contamination of RNA
preparations with ribonucleases (enzymes
that
degrade
RNA).
Ribonucleases
are
present in signi±cant amounts in a variety
of locations from which they may gain
access to samples of puri±ed RNA (e.g.
the
skin
surface,
saliva
aerosols
from
coughing or sneezing, and airborne dust).
Once a high quality RNA preparation has
been obtained from the appropriate tissue,
the presence of mRNA transcripts of the
transgene can be determined by RNA slot-
blot hybridization. A more informative
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