222
Animal Biotechnology and Modeling
cell transfer were used to transfer whole
genomes into mouse ova. From the early
1980s, beyond DNA microinjection, retro-
viral infection, ES cell technologies, and
nuclear transfer, we have also seen devel-
opment of cytoplast and karyoplast fusions
(including nuclear transfer), spermatozoa-
and spermatogonial cell–mediated trans-
fer, and particle bombardment and jet
injection procedures. The later techniques
expand upon earlier ‘‘building blocks’’ of
genetic engineering technologies. As such,
our capabilities and ef±ciencies will surely
continue to evolve. Yet, there have been
some conspicuous developments of gen-
eral interest to the scienti±c community
that may augment some of these exist-
ing methods.
Gain-of-function
and
loss-of-function
modeling have, for the most part, concen-
trated on introducing speci±c mutations
into the nuclear genome. From a gene
ablation standpoint, creation of loss-of-
function
models
will
be
facilitated
by
the emerging technology of RNA inter-
ference (RNAi). Short, interfering RNA
(siRNA) exists in a double-stranded state
and inhibits endogenous genes (and/or
exogenous sequences as in viral genes)
due to a complementary sequence ho-
mology. RNAi technology has potential
applications including the inhibition of vi-
ral gene transcription and inhibition of
endogenous genes coding for deleterious
gene products. Indeed, germ line com-
petent transgenic mouse and rat models
using RNA interference have been shown
to recapitulate null phenotypes.
In contrast to various methods to target
modi±cation of the nuclear genome, little
attention was focused until recently, on the
importance of mitochondrial genetics and
the mitochondrial genome in mammalian
development. This omission, in part, is
related to the dif±culty associated with
in
vivo
mitochondrial transfer. Without an
approach in hand, a signi±cant technolog-
ical hurdle remained in the identi±cation
of mitochondrial gene targets appropriate
for engineering or modi±cation. For a host
of applications, the ability to manipulate
the mitochondrial genome and to regu-
late mitochondrial gene function would
provide an additional target in modifying
mammalian development.
We and others have initiated studies re-
volving around mitochondrial transfer and
techniques to produce animals harboring
foreign mitochondrial genomes. The cre-
ation of transmitochondrial animals repre-
sents a new model system that will provide
a greater understanding of mitochondrial
dynamics, leading to the development of
genetically engineered production animals
as well as therapeutic strategies for human
metabolic diseases affected by mitochon-
drial mutation or function.
2.4
Other Laboratory Animal Models
Transgenic animal protocols developed in
mice have been modi±ed to accommodate
production of other transgenic species. In
particular, the larger body sizes of rats
and rabbits,
their short estrous cycles
and
gestation
lengths,
their
relatively
rapid generation times, and their litter-
bearing ability have helped make these
animals preferred models for several areas
of research employing gene transfer. As
with mice, DNA microinjection has been
t
h
em
e
t
h
o
du
s
e
dm
o
s
tf
r
e
q
u
e
n
t
l
yf
o
r
introduction of foreign DNA. Differences
between these species and mice in the size
of ova, physical response to microinjection,
requirements for
in vitro
ova culture, and
numbers of fertilized eggs needed for
embryo transfer (to maintain pregnancy),
as well as differences in general husbandry
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