234
Animal Biotechnology and Modeling
in that the activities of the enzymes can be
assayed, thus greatly increasing the sensi-
tivity of detection.
In analyzing the expression of any trans-
gene, it is always important to evaluate
at least two separate lines of transgenic
animals, to show that the expression pat-
terns are consistent and reproducible. It
is not uncommon for the site of inte-
gration within a given line of transgenic
animals to have profound influence on
transgene expression, independent of any
transcriptional control sequences in the
transgene itself.
5
Conclusions and Future Directions
5.1
Gene Transfer Today
The efforts and costs for gene-transfer
experimentation in animal biology are
challenging and limiting. Innovative tech-
nologies to enhance experimental gene-
transfer
efFciency
in
different
species
are desperately needed. Such enabling
techniques
would
not
only
bring
the
cost of individual projects into a rea-
sonable realm but would also increase
the likelihood of breakthrough studies
in many disciplines. As stated earlier,
aside from mouse modeling, DNA mi-
croinjection
into
preimplantation
em-
bryos
has
been
the
only
reproducible
means for heritable gene transfer in the
preponderance
of
mammalian
species,
other than multigene-targeted (nonspe-
ciFc) blastomere aggregation and nuclear
transplantation studies.
The outlined efFciencies for the pro-
duction of transgenic animals represent
what is considered to be current ‘‘state-of-
the-art’’ technology. It is envisioned that
procedures will be modiFed and enhanced
as new research efforts are reported. Under
the circumstances, there are a number of
speciFc achievements that would signiF-
cantly enhance experimental productivity.
Enabling technologies would center on the
following areas:
•
development of alternate DNA delivery
systems (e.g. embryonic stem cell trans-
fer for stable gene transfer, liposome-
mediated
gene
transfer,
or
perhaps
targeted somatic-cell techniques);
•
the identiFcation of optimal experimen-
tal conditions for a given form of gene
transfer and the identiFcation of ani-
mal strains best suited to the individual
technologies;
•
complete animal genome mapping and
identiFcation
of
homologies
to
hu-
man genes;
•
t
h
ee
s
t
a
b
l
i
s
hm
en
to
fr
o
u
t
in
ean
de
f
-
Fcient embryo and germ plasm cul-
ture systems;
•
the development of a means to reduce
the number of animals and embryos
required (e.g. use of PCR ampliFcation
of blastomere DNA or fluorescence-
activated cell sorter (±ACS) analyses and
gating of transfected germ plasm);
•
enhancement of relative experimental
efFciencies to minimize the numbers of
animals, gametes, and related reagents
necessary to generate founder animals
or experimental models.
Primary considerations for genetic ma-
nipulations
and gene
transfer
are
not
limited to donor and recipient require-
ments, surgical manipulations, embryo
handling,
and
gene-transfer skills,
but
also include expertise in tissue/cell cul-
ture and molecular biological techniques.
±or any animal experimentation, ethical
considerations, regulatory requirements,
