© The Nobel Committee for Physiology or
Medicine
illustration: Annika Rohi
Gene targeting is often used to inactivate
single genes. Such gene ‘knockout’ experiments
have
elucidated the roles of numerous genes in
embryonic development, adult physiology, aging
and
disease. To date, more than ten thousand mouse
genes (approximately half of the genes in the
mammalian genome) have been knocked out. Ongoing
international efforts will make ‘knockout mice’
for all genes available within the near future.
With gene targeting it is now possible to
produce almost any type of DNA modification in
the
mouse genome, allowing scientists to establish
the roles of individual genes in health and
disease. Gene targeting has already produced
more than five hundred different mouse models of
human disorders, including cardiovascular and
neuro-degenerative diseases, diabetes and
cancer.
Modification of genes by homologous
recombination
Information about the development and function
of our bodies throughout life is carried within
the DNA. Our DNA is packaged in chromosomes,
which occur in pairs – one inherited from the
father
and one from the mother. Exchange of DNA
sequences within such chromosome pairs increases
genetic
variation in the population and occurs by a
process called homologous recombination. This
process
is conserved throughout evolution and was
demonstrated in bacteria more than 50 years ago
by the
1958 Nobel Laureate Joshua Lederberg.
Mario Capecchi and Oliver Smithies both had the
vision that homologous recombination could be
used to specifically modify genes in mammalian
cells and they worked consistently towards this
goal.
Capecchi demonstrated that homologous
recombination could take place between
introduced DNA and
the chromosomes in mammalian cells. He showed
that defective genes could be repaired by
homologous recombination with the incoming DNA.
Smithies initially tried to repair mutated genes
in human cells. He thought that certain
inherited blood diseases could be treated by
correcting
the disease-causing mutations in bone marrow
stem cells. In these attempts Smithies
discovered
that endogenous genes could be targeted
irrespective of their activity. This suggested
that all
genes may be accessible to modification by
homologous recombination.
Embryonic stem cells – vehicles to the mouse
germ line
The cell types initially studied by Capecchi and
Smithies could not be used to create
gene-targeted animals. This required another
type of cell, one which could give rise to germ
cells. Only then could the DNA modifications be
inherited.
Martin Evans had worked with mouse embryonal
carcinoma (EC) cells, which although they came
from
tumors could give rise to almost any cell type.
He had the vision to use EC cells as vehicles to
introduce genetic material into the mouse germ
line. His attempts were initially unsuccessful
because EC cells carried abnormal chromosomes
and could not therefore contribute to germ cell
formation. Looking for alternatives Evans
discovered that chromosomally normal cell
cultures
could be established directly from early mouse
embryos. These cells are now referred to as
embryonic stem (ES) cells.
The next step was to show that ES cells could
contribute to the germ line. Embryos from one
mouse
strain were injected with ES cells from another
mouse strain. These mosaic embryos (i.e.
composed of cells from both strains) were then
carried to term by surrogate mothers. The mosaic
offspring was subsequently mated, and the
presence of ES cell-derived genes detected in
the pups.
These genes would now be inherited according to
Mendel’s laws.
Evans now began to modify the ES cells
genetically and for this purpose chose
retroviruses, which
integrate their genes into the chromosomes. He
demonstrated transfer of such retroviral DNA
from
ES cells, through mosaic mice, into the mouse
germ line. Evans had used the ES cells to
generate
mice that carried new genetic material.
Two ideas come together – homologous
recombination in ES cells
By 1986 all the pieces were at hand to begin
generating the first gene targeted ES cells.
Capecchi and Smithies had demonstrated that
genes could be targeted by homologous
recombination
in cultured cells, and Evans had contributed the
necessary vehicle to the mouse germ line – the
ES-cells. The next step was to combine the two.
For their initial experiments both Smithies and
Capecchi chose a gene (hprt) that was easily
identified. This gene is involved in a rare
inherited human disease (Lesch-Nyhan syndrome).
Capecchi refined the strategies for targeting
genes and developed a new method
(positive-negative
selection, see Figure) that could be generally
applied.
Birth of the knockout mouse – the beginning
of a new era in genetics
The first reports in which homologous
recombination in ES cells was used to generate
gene-targeted mice were published in 1989. Since
then, the number of reported knockout mouse
strains has risen exponentially. Gene targeting
has developed into a highly versatile
technology.
It is now possible to introduce mutations that
can be activated at specific time points, or in
specific cells or organs, both during
development and in the adult animal.
Gene targeting is used to study health and
disease
Almost every aspect of mammalian physiology can
be studied by gene targeting. We have
consequently witnessed an explosion of research
activities applying the technology. Gene
targeting has now been used by so many research
groups and in so many contexts that it is
impossible to make a brief summary of the
results. Some of the later contributions of this
year’s
Nobel Laureates are presented in the following
page.
Gene targeting has helped us understand the
roles of many hundreds of genes in mammalian
fetal
development. Capecchis research has uncovered
the roles of genes involved in mammalian organ
development and in the establishment of the body
plan. His work has shed light on the causes of
several human inborn malformations.
Evans applied gene targeting to develop mouse
models for human diseases. He developed several
models for the inherited human disease cystic
fibrosis and has used these models to study
disease
mechanisms and to test the effects of gene
therapy.
Smithies also used gene targeting to develop
mouse models for inherited diseases such as
cystic
fibrosis and the blood disease thalassemia. He
has also developed numerous mouse models for
common human diseases such as hypertension and
atherosclerosis.
In summary, gene targeting in mice has pervaded
all fields of biomedicine. Its impact on the
understanding of gene function and its benefits
to mankind will continue to increase over many
years to come.
Mario R. Capecchi, born 1937 in Italy, US
citizen, PhD in Biophysics 1967, Harvard
University, Cambridge, MA, USA. Howard Hughes
Medical Institute Investigator and Distinguished
Professor of Human Genetics and Biology at the
University of Utah, Salt Lake City, UT, USA.
Sir Martin J. Evans, born 1941 in Great
Britain, British citizen, PhD in Anatomy and
Embryology 1969, University College, London, UK.
Director of the School of Biosciences and
Professor of Mammalian Genetics, Cardiff
University, UK.
Oliver Smithies, born 1925 in Great
Britain, US citizen, PhD in Biochemistry 1951,
Oxford
University, UK. Excellence Professor of
Pathology and Laboratory Medicine, University of
North
Carolina at Chapel Hill, NC, USA.
Source: Medindia