Biomedical Engineering Reference
In-Depth Information
of this system for the analysis of gene expression and
function. Some of the DNA does become incor-
porated into the genome and may be transmitted
through the germ line (Rusconi and Schaffner
1981). However, integration occurs at a very low
frequency and, given the long generation interval
of
Xenopus laevis
(12-18 months from egg to
adult), this is not an efficient way to generate trans-
genic frogs.
A simple and efficient process for large-scale
transgenesis in
Xenopus
has become available only
in the last few years (Kroll & Amaya 1996). In this
technique, known as restriction-enzyme-mediated
integration (REMI), linearized plasmids containing
the transgene of interest are mixed with decon-
densed sperm nuclei and treated with limiting
amounts of a restriction enzyme to introduce nicks
in the DNA. The nuclei are then transplanted into
unfertilized
Xenopus
eggs, where the DNA is repaired,
resulting in the integration of plasmid DNA into the
genome. This technique allows the production of up
to 700 transgenic embryos per person per day, most
of which survive at least to the swimming-tadpole
stage. The decondensed nuclei are extremely fragile,
so careful handling and transplantation within
about 30 min are required for a good yield of normal
transgenic embryos. In some cases, viable trans-
genic adults have been derived from the tadpoles
and transgenic
X. laevis
lines have been established
(Bronchain
et al
. 1999, Marsh-Armstrong
et al
.
1999). A disadvantage of
X. laevis
is that the species
is tetraploid. Offield
et al
. (2000) have therefore
established transgenic lines of the closely related but
diploid species
Xenopus tropicalis
, which also has a
shorter generation interval than its tetraploid cousin.
Since
Xenopus
is used worldwide as a develop-
mental model organism, transgenic
Xenopus
techno-
logy has been rapidly adopted in many laboratories
and is being used to examine (or in many cases
re-examine) the roles of developmental genes. Thus
far, the sophisticated tools used in transgenic mice
have not been applied to
Xenopus
, but this is only a
matter of time. Recently, an inducible expression
system based on the use of a
Xenopus
heat-shock
promoter was described, allowing inducible control
of the GFP gene. This system has been used to invest-
igate Wnt signalling in early
Xenopus
development
(Wheeler
et al
. 2000). As discussed above, one of the
early successes in transgenic mouse methodology
was the expression of rat growth hormone, resulting
in transgenic mice up to twice the size of their non-
transgenic siblings. The role of growth hormone in
amphibian metamorphosis has now been examined
by expressing
Xenopus
growth hormone in trans-
genic frogs. The transgenic tadpoles developed at the
same rate as control tadpoles, but typically grew to
twice the normal size (Huang & Brown 2000). After
metamorphosis, the transgenic frogs also grew much
more quickly than controls and showed skeletal
defects.
Gene transfer to fish
Fish transgenesis can be used to study gene function
and regulation, e.g. in model species, such as the
zebrafish (
Danio rerio
) and medaka (
Oryzias latipes
),
and to improve the traits of commercially important
species, such as salmon and trout. Gene-transfer
technology in fish has lagged behind that of mam-
mals, predominantly due to the lack of suitable
regulatory elements to control transgene expression.
The first transgenic fish carried transgenes driven
by mammalian or viral regulatory elements, and
their performance varied considerably. For example,
attempts to express growth-hormone genes in trout
initially met with little success, and this may have
been due to the inability of fish cells to correctly pro-
cess mammalian introns (Betancourt
et al
. 1993).
However, fish are advantageous assay systems for
several reasons, including their fecundity, the fact
that fertilization and development are external and
the ease with which haploid and uniparental diploid
embryos can be produced (Ihssen
et al
. 1990).
Like frogs, the injection of DNA into fish eggs and
early embryos leads to extensive replication and
expression from unintegrated transgenes, so that
fish, like frogs, can be used for transient expression
assays (Vielkind 1992). Some of the DNA integrates
into the genome, leading to germ-line transmission
and the production of transgenic fish lines (reviewed
by Iyengar
et al
. 1996). There has been recent
progress in the development of transgenic fish
with enhanced growth characteristics, particularly
through the use of expression constructs that are
derived from the same species (e.g. Rahman
et al
.
1998; reviewed by Dunham 1999). It is likely that
