Brain Development
known to be activated through dimer
formations, and if truncated forms are
expressed, normal receptor functions are
effectively perturbed (Fig. 2a). In the
system, double-stranded RNA se-
quences, which are simply designed from
the coding sequences of a gene of inter-
est, have been demonstrated to speci±cally
interfere with mRNA transcription and/or
translation of the gene. This mechanism
is now shown to be appli-
cable not only to invertebrates but also to
vertebrates including the mouse. Apply-
ing the
antisense oligonucelotides
for a given
gene sequence, has also been revealed to
block mRNA translation. Most recently,
it has been demonstrated that the
pholino oligonucleotides
serve as the best
tool to stably block gene functions because
the morpholino reagents are not degraded,
thereby inhibiting mRNA translation for a
long time.
Generating an organism that completely
lacks a gene of interest is a prevailing
method, and the manipulated organism
could yield more reproducible and re-
liable results. In the mouse, embryonic
stem cells (ES) that can differentiate into
all cell types in an organism have been
established, and it is now a routine pro-
cess to replace a gene of interest with a
drug selection gene in the ES cells via
homologous recombination. Selected ES
cells can directly be injected in a host em-
bryo, and chimeric mice containing cells
derived from the gene manipulated ES
cells are easily obtained. If ES cell–derived
cells contribute to the germ line, mice ho-
mozygous for the mutated locus can be
±nally produced by means of serial mat-
ing processes (Fig. 11). This procedure,
gene targeting
gene knockout
greatly contributed to the understanding of
the unveiled functions of genes expressed
in the mouse brain. Major problems in
loss-of-function studies are that redun-
dant functions of related genes sometimes
mask the mutant phenotypes and that
the abnormalities at earlier developmental
stages and/or lethality prevent the mutant
phenotypes from being examined at later
stages. The former problem might sim-
ply be overcome by generating double or
more related gene knockouts, while the
latter could partly be solved by using the
conditional gene knockout system that al-
lows the gene of interest to be inactivated
in a spatiotemporally restricted manner.
Gain-of-function Studies
To express a gene of interest ectopically,
ubiquitous promoters such as heat shock,
beta actin, cytomegalovirus (CMV) pro-
moters, and so on must be linked to the
gene, and the construct should be delivered
into tissues or organisms. If a tissue or cell
type speci±c overexpression is required,
the researchers could use proper types of
promoters. For example, the promoter of
neuron-speci±c enolase is widely used to
drive the neuron-speci±c expression of ex-
ogenous genes. Synthesized mRNA with
cap can also be injected into a fertil-
ized egg or the blastomere of a developing
embryo, and strong expression from the
injected mRNA has been reported in the
zebra±sh and frog systems. In the mouse,
the constructs for overexpression can be
easily microinjected into the pronuclei of
fertilized eggs, and mice lines with the ex-
ogenous gene will be obtained after oviduct
transfer of the injected eggs.
Recently, the EP mediated gene trans-
fer method into living embryos and/or
tissues has emerged a powerful tool. Us-
ing controlled square pulses instead of
authentic pulses with exponential decay,
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