Sat. Jan 22nd, 2022

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© 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

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