Researchers at the RIKEN Brain Science Institute are
developing a highly efficient system to create mouse model lines
that could dramatically advance genetic research on the most common
mental retardation.
 |
| Laboratory Head Kazuhiro Yamakawa (middle) and members of the
Nurogenetics Laboratory including four members participating in the
Down syndrome project. Other members of the laboratory are involved
in epilepsy research. |
| Copyright : RIKEN 2010 |
Chromosome 21 is the smallest of the 23 pairs of chromosomes in
humans, yet it is responsible for Down syndrome—the most
common genetic mental retardation. Down syndrome (DS) is caused by
the erroneous replication of human chromosome 21 (HC21), which
results in three copies of the chromosome instead of the normal
pair of two. The most common cause of this ‘trisomy’ on
HC21 is the failure of the chromosome pair to divide in an egg
cell—often linked to advanced maternal age. Such an egg cell
has two copies of HC21, and when fertilized, accepts another copy
of HC21 from the sperm cell, resulting in a total of three, instead
of the normal two, copies of the chromosome.
Unlocking the molecular pathology of trisomy 21 is greatly
anticipated. Worldwide, it is estimated that up to one in every 700
babies is born with DS, and there are no specific therapeutic
treatments. Yet little is known as to what determines the various
phenotypes associated with the disorder. Patients typically suffer
from neurological and behavioral difficulties, including language
delays and attention difficulties, and some also face
immunological, digestive and cardiac problems. The severity of
mental retardation differs by patient and age—sometimes the
symptoms are alleviated with age, while for others the symptoms
become worse, developing into conditions such as Alzheimer’s
disease. “We’d like to understand the molecular
pathways responsible for the disease so that we can contribute to
the development of effective therapies in the future,” says
Kazuhiro Yamakawa, head of the Neurogenetics Laboratory at the
RIKEN Brain Science Institute in Wako, Saitama.
In fiscal 2008, Yamakawa’s team secured a grant from the
President’s Fund under the category of ‘challenging
research’ for a two-year project aimed at developing a highly
efficient system to generate transgenic mouse lines for DS
research. Their goal is to establish a high-throughput system to
generate partial-trisomic DS mouse models and to identify the gene
or genes responsible for DS features. In the 14-member laboratory,
which also studies epilepsy, Yamakawa and four young researchers
participate in this exciting project.
Down syndrome mouse models and lines of research
HC21 carries approximately 360 genes and contains the Down syndrome
critical region (DSCR) that many researchers believe is key to the
occurrence of DS. HC21 is orthologous (similar by shared ancestry)
to part of mouse chromosome 16 (MC16), and several fortuitously
generated DS mouse models with partial MC16 have been reported,
including Ts1Cje, which has approximately 100 genes and is
responsible for milder learning defects; and Ts2Cje, which has
approximately 140 genes, partially shared with Ts1Cje, and is
responsible for learning defects. Both Ts1Cje and Ts2Cje trisomic
segments contain DSCR.
Some researchers believe that the imbalance in chromosomal number
for HC21 induces DS by affecting the expressions of genes on the
overall genome. Yamakawa and several other groups, however, support
a different idea that the initial switch for the disorder is the
dosage-dependent overexpression of genes located only on HC21. In
2004, Yamakawa’s team found that the expression levels of
genes in the trisomic region of MC16 in Ts1Cje mice were increased
by 50%, whereas the levels of other genes on other chromosomes or
the normal euploid region of MC16 were almost the same as in normal
mice1. Yamakawa adds that many of these overexpressed genes may not
result in substantive damage, so identification of a few
‘master’ genes is critical. Some of those genes might
also work collectively to contribute to the disorder.
In attempts to identify such master genes, several transgenic mouse
models overexpressing individual candidate genes in distinct
systems have been developed in other laboratories. However, even
though some of the mice models displayed DS-like phenotypes, such
as learning disability, the expressions of HC21 or MC16 genes in
those models were too excessive (much more than 1.5-fold), ectopic
(out of place) or occurred in the wrong developmental timings,
making it difficult to compare the results. To improve on this,
Yamakawa and his colleagues established a common platform in which
heterozygous knockout mice for the HC21-orthologous MC16 candidate
gene were mated with partial trisomy MC16 mice such as Ts1Cje. This
procedure ensures that the number of copies of the candidate gene
is returned to two, while other genes on the trisomic segment
retain three copies. Using this system, it is possible to
investigate whether any of the DS-like abnormal phenotypes are
improved, allowing the contribution of each gene to the
biochemical, biophysical or behavioral DS abnormal parameters to be
compared impartially. The team has already identified and reported
a large number of parameter abnormalities, including decreases in
mitochondrial membrane potential and adenosine triphosphate
production, increases in reactive oxygen species and kinase
activities in Ts1Cje, and enlarged brain ventricles, impaired
developmental and adult neurogenesis in Ts1Cje and Ts2Cje. These DS
mouse models also display behavioral abnormalities indicative of
learning defects. Knockout mice for more than ten HC21-orthologous
MC16 genes have been generated, including a model for Dscam—a
neural cell adhesion molecule that Yamakawa’s team has long
been investigating as a promising candidate for DS mental
retardation2. The team is now characterizing mice obtained by
mating these model mice with Ts1Cje or Ts2Cje, and evaluating the
role of each gene in the DS-like abnormal phenotypes.
A highly efficient system for generating Down syndrome mouse
models
Although Yamakawa’s strategy is promising, the generation of
many knockout mice is still a daunting task. To implement
large-scale analysis more effectively, Yamakawa's group is now
establishing a new system that will allow mice to be generated with
high efficiency and with free design of partial trisomic segments
harboring gene knockouts as desired. In their approach, a selection
marker is introduced into MC16 in a mouse primary fibroblast, which
is then processed into ‘microcells’, each of which
contains, on average, a single chromosome string fused to a special
‘X’ cell. In the cells containing MC16, which are
separated out, MC16 can be manipulated to achieve the desired
chromosomal segments and gene knockouts with high efficiency. The
designed cell containing recombinant MC16 is once again processed
into microcells, and then fused into a mouse embryonic stem cell to
generate a partial trisomic MC16 mouse. “This system will
enable us to not only accelerate our research but also implement
experiments that have been impossible, such as the inactivation of
multiple genes simultaneously,” Yamakawa says.
Atsushi Shimohata, a member of the technical staff at
Yamakawa’s laboratory, and Kenji Amano, a research associate,
are currently devoting much of their endeavors to establishing this
new system and to removing unnecessary segments from MC16 within
the fused cell. Ei-ichi Takaki and Sachie Asada, both research
scientists in Yamakawa’s laboratory, are concurrently
attempting to establish backup systems, including another type of
mouse generating system and a high-efficiency in vitro screening
system.
Within 3–5 years, Yamakawa hopes to establish these systems
and identify several critical DS genes to elevate DS research to
the next level. “We hope that our great passion for this
project will eventually lead to alleviating patients’
conditions,” Yamakawa says. |
 |
| Mouse models with partial trisomy 16, such as Ts2Cje and
Ts1Cje, are widely studied to identify the genes responsible for
Down syndrome. |
| Copyright : RIKEN 2010 |
|
 |
| A flow for the generation of ideal DS model mice having freely
designed segments of MC16 as partial trisomic segments. |
| Copyright : RIKEN 2010 |
|
|
Journal information
1. Amano, K., Sago, H., Uchikawa, C., Suzuki T., Kotliarova, E.
S., Nukina N., Epstein J. C. & Yamakawa, K. Dosage-dependent
over-expression of genes in the trisomic region of Ts1Cje mouse
model for Down syndrome. Human Molecular Genetics 13,
1333–1340 (2004).
2. Amano, K., Fujii, M., Arata, S., Tojima, T., Ogawa, M., Morita,
N., Shimohata, A., Furuichi, T., Itohara, S., Kamiguchi, H.,
Korenberg, J. R., Arata, A. & Yamakawa, K. DSCAM deficiency
causes loss of pre-inspiratory neuron synchroneity and perinatal
death. Journal of Neuroscience 29, 2984–2996 (2009).
SOURCE