Biomedical Engineering Reference
In-Depth Information
Wild type mouse
shaker-2
mouse
Genomic DNA
BAC
genomic
library
Homozygous
shaker-2
eggs
Inject individual
BAC clones
BAC clones
spanning
shaker-2
candidate region
Fig. 6.16
Functional
complementation in transgenic
mice to isolate the
Shaker-2
gene.
Homozygous
shaker-2
fertilized mouse
eggs were injected with BAC clones
derived from the
Shaker-2
candidate
region of a wild-type mouse. Progeny
were screened for restoration of the
wild-type phenotype, thus identifying
the BAC clone corresponding to the
Shaker-2
gene. This clone is then
sequenced and used to isolate and map
the corresponding human disease
gene
DFNB3
.
Sequence
Identify clone
that corrected defect
Screen human library
Map of human gene
quite possible to carry out functional cloning of, for
example, mammalian proteins in bacteria and yeast.
Thus, complementation in yeast has been used to
isolate cDNAs for a number of mammalian meta-
bolic enzymes (e.g. Botstein & Fink 1988) and certain
highly conserved transcription factors (e.g. Becker
et al
. 1991), as well as regulators of meiosis in plants
(Hirayama
et al
. 1997). This approach can also
be used in mammalian cells, as demonstrated by
Strathdee
et al
. (1992), who succeeded in isolating
the
FACC
gene, corresponding to complementation
group C of Fanconi's anaemia. Generally a pool sys-
tem is employed, where cells are transfected with a
complex mix of up to 100 000 clones. Pools that
successfully complement the mutant phenotype are
then subdivided for a further round of transfection,
and the procedure repeated until the individual
cDNA responsible is isolated.
Functional complementation is also possible in
transgenic animals and plants. In this way, Probst
et al
. (1998) were able to clone the mouse deafness-
associated gene
Shaker-2
, and from there its human
homologue,
DFNB3
(Fig. 6.16). The
shaker-2
muta-
tion had previously been mapped to a region of
the mouse genome that is syntenic to the region
involved in a human deafness disorder. BAC clones
corresponding to this region were therefore prepared
from wild-type mice and microinjected into the eggs
of
shaker-2
mutants. The resulting transgenic mice
were screened for restoration of a normal hearing
