Animal Biotechnology and Modeling
apparatus to physically inject the DNA
construct solution into the male pronu-
clei or nuclei of fertilized one- or two-cell
ova. As such, the method is also sometimes
referred to as
pronuclear microinjection
Virtually any cloned DNA construct can
be used, albeit some caveats apply. Lin-
earized (as opposed to closed, circular)
DNA constructs are more readily inte-
grated into the host genome. The presence
of extraneous plasmid- or vector-related
DNA sequences associated with the con-
struct can adversely affect expression of
the integrated construct/transgene. Also,
greater care must be taken when handling
larger DNA constructs, such as those de-
rived from yeast artiFcial chromosomes
(YACs), to avoid damage by shearing prior
to and during microinjection.
With few exceptions, microinjected con-
structs integrate randomly throughout the
host’s genome, but usually only in sin-
gle locations. This fact can be exploited to
obtain functional linkage of independent
transgenes by simultaneous coinjection of
more than one DNA construct. In such
a case, both constructs tend to integrate
in the same randomly located site and
may exhibit coexpression. Because integra-
tion is spatially random, transgenes may
be integrated into either a somatic or a
sex chromosome.
The integration process itself is poorly
understood, but it apparently does not
involve homologous recombination. Dur-
ing integration, single or multiple copies
(1–200) of a transgene are incorporated
into the genomic DNA, predominantly as
head-to-tail concatemers. Regulatory ele-
ments in the host DNA near the site
of integration, and the general availabil-
ity of this region for transcription, appear
to play a major role in affecting the level
of transgene expression. This ‘‘positional
effect’’ is presumed to explain why the lev-
els of expression of the same transgene
founder animals as well as their offspring
(or ‘‘lines’’). It is therefore prudent to
examine transgene expression in offspring
from at least three or four founder animals
to determine what might be a result of
position or incorporation site influences.
Additionally, the presence of tissue- or cell-
type-speciFc nuclear regulatory factors that
act on host genes near the site of integra-
tion in cis or trans may restrict transgene
expression to only a subpopulation of cells
even though the transgene is present in
virtually all cells of a founder.
tion frequently undergoes various forms
of sequence duplication, deletion, or rear-
rangement as a result of transgene incor-
poration. Such alterations, if sufFciently
drastic, may disrupt the function of nor-
mally active host genes at the integration
site, an activity that constitutes ‘‘insertional
mutagenesis,’’ wherein an aberrant phe-
notype may result. Such events cannot be
purposefully designed, but they have led to
the serendipitous discovery of previously
unsuspected genes and gene functions.
Because gene transfer is accomplished
at the one- to two-cell stage very early
in embryonic development, the transgene
is incorporated into essentially every cell
that contributes to development of the
‘‘founders’’) produced by this method are
considered to
or ‘‘nonchimeric,’’ in the sense that the
nuclei of all cells of the body. The DNA
or gene construct must be incorporated
into the one-cell ovum (or a single viable
blastomere) prior to DNA replication in
order to appear in every descendent cell.
If incorporation or integration occurs at
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