Biomedical Engineering Reference
In-Depth Information
1995a,b,c, Soriano 1995, Muller 1999) and a num-
ber of Internet databases have been established to
keep track of the results (see Sikorski & Peters 1997).
The phenotypes of homozygous, null mutant mice
provide important clues to the normal function of
the gene. Some gene knockouts have resulted in
surprisingly little phenotypic effect, much less severe
than might have been expected. For example,
myoD
,
whose expression in transfected fibroblasts causes
them to differentiate into muscle cells, and which
was therefore a good candidate as a key regulator of
myogenesis, is not necessary for development of a
viable animal (Rudnicki
et al
. 1992). Similarly, the
retinoic acid
resulting in the production of humanized antibodies
in transgenic mice (Moore
et al
. 1995). The Cre-
loxP
site-specific recombinase system has been used exten-
sively in ES cells to generate mice in which condi-
tional or inducible gene targeting is possible and to
produce defined chromosome deletions and trans-
locations as models for human disease. We shall
discuss the many applications of Cre-
loxP
and other
site-specific recombinase systems in Chapter 13.
Other mammals and birds
Traditional techniques
receptor is not necessary for viable
mouse development in knockout mice (Lohnes
et al
.
1993), even though this receptor is a necessary
component of the pathway for signalling by retinoids
and has a pattern of expression quite distinct from
other retinoic acid receptors in embryos. Such ob-
servations have prompted speculation that genetic
redundancy may be common in development, and
may include compensatory up-regulation of some
members of a gene family when one member is
inactivated. An example of this may be the up-
regulation of
myf-5
in mice lacking
myoD
(Rudnicki
et al
. 1992). Gene knockouts have also been used as
mouse models of human diseases, such as cystic
fibrosis,
γ
The three major routes for producing transgenic mice
have also been used in other mammals and birds,
particularly in farm animals. The efficiency of each
procedure is much lower than in mice. Pronuclear
microinjection in mammals such as sheep and cows,
for example, typically results in less than 1% of the
injected eggs giving rise to transgenic animals. Added
to this, the recovery of eggs from donor animals and
the reimplantation of transformed eggs into foster-
mothers is a less efficient procedure and requires, at
great expense, a large number of donors and reci-
pients. The eggs themselves are also more difficult to
manipulate - they are very delicate and tend to be
opaque. It is often necessary to centrifuge the eggs in
order to see the pronuclei. In chickens, it is possible
to remove eggs just after fertilization and microinject
DNA into the cytoplasm of the germinal disc, where
the male and female pronuclei are to be found.
However, it is not possible to return the manipulated
eggs to a surrogate mother, so they must be cultured
in vitro
. Using this procedure, Love
et al
. (1994)
obtained seven chicks, equivalent to about 5% of
the eggs injected, that survived to sexual maturity.
One cockerel transmitted the transgene to a small
proportion of his offspring, indicating that he was
chimeric for transgene integration.
The use of retroviruses to produce transgenic
chickens has been reported by Bosselman
et al
.
(1989). These investigators injected a replication-
defective recombinant reticuloendotheliosis virus
carrying the
neo
gene into laid eggs and found
that approximately 8% of male birds carried vector
sequences. The transgene was transmitted through
the germ line in a proportion of these birds and
-thalassaemia and fragile X syndrome
(reviewed by Bedell
et al
. 1997; see Chapter 14).
While most gene-targeting experiments in mice
have been used to introduce mutations into genes
(either disruptive insertional mutations or subtle
changes), the scope of the technique is much wider.
The early gene-targeting experiments demonstrated
that this approach could also be used to correct
mutated genes, with obvious applications in gene
therapy. Homologous recombination has also been
used to exchange the coding region of one gene for
that of another, a strategy described as 'gene knock-
in'. This has been used, for example, to test the
ability of the transcription factors Engrailed-1 and
Engrailed-2 to compensate for each other's functions.
Hanks
et al
. (1995) replaced the coding region of the
engrailed-1
gene with that of
engrailed-2
, and showed
that the
engrailed-1
mutant phenotype could be
rescued. A more applied use of gene knock-in is
the replacement of parts of the murine immuno-
globulin genes with their human counterparts,
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