Animal Biotechnology and Modeling
225
identify and characterize transgenic pigs
and
illustrate
the
extended
time
lines
associated with the production of any
transgenic livestock model. In addition,
t
h
et
im
ef
r
am
ef
r
omb
i
r
t
ho
fp
i
g
so
r
cattle to the establishment of lines can be
1–2 years to 4–5 years respectively (while
also dependent on the sex of founders).
Hence, there is an obvious advantage to
characterizing transgenic mouse models
to
expedite
what
will
ultimately
be
a
lengthy undertaking.
3.2.2
Retroviral Infection
Retroviruses have been used as vectors for
transfer of foreign genes into domestic ani-
mal genomes, and are particularly exciting
because of the promise of 100% transfer ef-
Fciency. Although embryos can be infected
with retroviruses up to midgestation, early
eggs, from oocytes to 16-cell stage ova, are
used for infection with one or more recom-
binant retroviruses containing a foreign
gene. Immediately following infection, the
retrovirus produces a DNA copy of its RNA
genome using the viral enzyme, reverse
transcriptase. Completion of this process
requires
that
the
host
cell
undergoes
the S-phase of the cell cycle. Therefore,
retroviruses effectively transduce only mi-
totically active cells. ModiFcations to the
retrovirus frequently consist of removal
of structural genes, such as
gag, pol
,and
env
, which support viral particle formation.
Additionally, most retroviruses and com-
plementary lines are ecotropic in that they
infect species-speciFc cell lines, limiting
risk to humans in animal experimentation.
Advantages of retrovirus-mediated gene
transfer include the technical ease and
efFciency of existing methods. Very high
rates of gene-transfer are achieved with
the use of retroviruses, thus, minimizing
the numbers of ova and animals needed
for
gene-transfer
studies.
Additionally,
integration of viral genes does not appear
to induce genetic rearrangements of the
host genome (occasionally reported with
other methods), with the exception of a
possible short duplication at the site of
integration.
However, of the disadvantages listed
in Sect. 2, the Frst two limitations ap-
pear
to
raise
the
greatest
concern
at
this time. ±rom a size perspective, the
maximum permissible size for efFcient en-
capsidation or reverse transcription of each
retroviral vector is approximately 10 kb.
Because introns of foreign genes carried
by retrovirus vectors are spliced out dur-
ing replication, the insert does not have
to contain introns, yet, intronic sequences
can be critical for optimal gene expression
and regulation. ±rom a biosafety perspec-
tive, disabled retroviruses may become
replication-competent via potential recom-
bination events that may occur between
or within retroviral vector, packaging cell
lines, and the host genome. Biocontain-
ment and safety considerations are still de-
bated. Yet, work to date has illustrated and
further characterized the utility of sensitive
analytical methods to detect unexpected vi-
ral transmission and recombination risks.
In addition to legislative and regulatory
oversight, conscientious mechanisms now
inp
lacehe
lpensuretha
tproperme
thods
and safeguards are incorporated into on-
going experimentation.
3.2.3
Nuclear Transfer
Cloning is generally associated with nu-
clear transfer whereby a nucleus provided
by a donor cell is introduced into an enucle-
ated oocyte (unfertilized ovum) or zygote
allowing for reprogramming of the de-
veloping embryo. It should be noted that
nuclear transfer, with nuclei obtained from
either mammalian stem cells or differenti-
ated adult cells, is an especially important
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