218
Animal Biotechnology and Modeling
any
later
time
point,
then
mosaicism
can occur. Incorporation of the transgene
into cells that will eventually contribute
to development of germ cells (sperm or
ova) is a common occurrence with this
method, and it makes possible heritability
of the transgene by offspring of founder
animals. In such cases, the transgene
has been said to have gone ‘‘germ-line.’’
However, integration of the microinjected
DNA construct into the host’s genome
occasionally may be inexplicably delayed.
In such a case, if cells of the early embryo
(‘‘blastomeres’’) undergo mitosis before
integration occurs, some but not all of the
cells will contain the transgene, and the
founder animal, although still considered
to be transgenic, will be classiFed as a
mosaic or chimera.
Advantages
of
the
microinjection
method are as follows: (1) the relatively
high frequency of generating transgenic
animals
from
viable
microinjected
embryos transferred to recipient females
(20–30%), (2) the high probability of germ-
line transmission of the transgene, (3) the
relative lack of constraints on the size
or type of DNA construct used, (4) the
relative stability of the transgene as it is
transmitted from generation to generation,
and (5) the low frequency of mosaicism or
double integrations (combined estimate of
10–30% of founders).
D
i
s
ad
v
an
t
ag
e
so
fth
i
sm
e
thodin
c
lud
e
the following: (1) the random and po-
tentially signiFcant influence of the site
of integration on transgene expression
(positional effects), (2) the potential for
undesired insertional mutagenesis, (3) the
occasional production of mosaic founders,
(4) the occasional lack of germ line in-
corporation, and (5) the time and expense
required to obtain the skills necessary for
micromanipulation and microinjection.
2.3.2
Retroviral Introduction of
Transgenes
Transfer of foreign genes into animal
genomes
has
also
been
accomplished
using retroviruses. Although embryos can
be infected with retroviruses up to midges-
tation, early eggs, usually at the 4- to
16-cell stages, are used for infection with
one or more recombinant retroviruses
containing a foreign gene. Immediately
following the infection, the retrovirus pro-
du
c
e
saDNAcop
yo
fi
t
sRNAg
enom
e
using the viral enzyme reverse transcrip-
tase. Completion of this process requires
theho
s
tce
l
ltounde
rgotheSpha
seo
f
the cell cycle. Therefore, retroviruses ef-
fectively transduce only mitotically active
cells. Incomplete infections, in which not
all embryonic cells acquire the retrovirus,
occur more frequently when using em-
bryos after the 4-cell stage, with resultant
chimeric embryos. 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 only rodents (e.g. rats, mice) and
rodent cell lines rather than humans.
T
h
eDNAc
o
p
yo
ft
h
ev
i
r
a
lg
en
om
e
,
or provirus, integrates randomly into the
host cell genome, usually without deletions
or rearrangements. However, as is the
case for gene transfer by microinjection,
because
integration
is
not
by
way
of
homologous recombination, this method
is not used effectively for site-directed
mutagenesis.
Very high rates of gene transfer are
achieved with
the
use
of retroviruses.
However, disadvantages include (1) a gen-
eral limitation on the size of the foreign
DNA insert that can be accommodated
by individual viral strains, (2) biosafety
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