Nobel Prize in Physiology or Medicine 2007: Gene Targeting in Mice Using Embryonic Stem Cells

This note covers the Nobel Prize in Physiology or Medicine 2007: who won it, how the three laureates learned to make precise, heritable changes to single genes in mice using embryonic stem cells, how "knockout mice" are built, how the discovery unfolded between the 1950s and 1989, why gene targeting matters for medicine, and quick facts for exams.

What was the Nobel Prize in Physiology or Medicine 2007 awarded for?

The Nobel Prize in Physiology or Medicine 2007 was awarded "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells", jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies.

In plain words, the three scientists worked out how to change one chosen gene inside a mouse, in a precise and controlled way, and have that change passed down to the mouse's offspring.

Before their work, scientists could insert extra genetic material into animals, but they could not reliably aim at a single existing gene and switch it off or alter it exactly where they wanted.

This combined technique became known as gene targeting, and mice carrying a deliberately disabled gene are called "knockout mice".

The official body that decides this prize is the Nobel Assembly at Karolinska Institutet, and the prize is formally called the Nobel Prize in Physiology or Medicine.

Who are the laureates?

All three laureates shared the prize equally, each receiving one third of the award.

Mario R. Capecchi

Mario R. Capecchi was born on 6 October 1937 in Verona, Italy. At the time of the award he was affiliated with the University of Utah, Salt Lake City, UT, USA, and with the Howard Hughes Medical Institute, USA. He held one third of the prize.

Capecchi showed that DNA placed into the nucleus of a mouse cell could become part of the cell's own genes by a process called homologous recombination.

He later developed a refined method, positive-negative selection, that made it possible to pick out, from millions of cells, the rare ones in which the targeting had worked correctly.

Sir Martin J. Evans

Sir Martin J. Evans was born on 1 January 1941 in Stroud, United Kingdom. At the time of the award he was affiliated with Cardiff University, Cardiff, United Kingdom. He held one third of the prize.

Evans identified and isolated embryonic stem cells (ES cells) from early mouse embryos, grew them in culture, and showed that genetic material carried in these cells could be passed into a living mouse and then inherited by its offspring.

This gave scientists a "vehicle" for carrying any chosen gene change into a whole animal.

Oliver Smithies

Oliver Smithies was born on 23 June 1925 in Halifax, United Kingdom, and died on 10 January 2017 in Chapel Hill, NC, USA. At the time of the award he was affiliated with the University of North Carolina, Chapel Hill, NC, USA. He held one third of the prize.

While trying to find ways to repair inherited blood diseases, Smithies discovered that a chosen gene in a cell could be altered by homologous recombination whether or not that gene was switched on at the time. This suggested that, in principle, almost any gene could be targeted for modification.

What problem were the laureates trying to solve?

Every cell in the body carries the same complete set of genetic instructions, written in DNA and packaged into chromosomes, which occur in pairs, one inherited from each parent. Making a deliberate, inheritable change to one gene is, according to the prize committee, a "two-part problem".

The first part is finding one specific stretch of DNA among the roughly three billion base pairs of the genome and altering it exactly as intended, rather than inserting new DNA at a random spot.

The second part is making sure that change reaches every cell of a new animal, including its reproductive cells, so that it can be passed on to the next generation according to Mendel's laws.

Capecchi and Smithies solved the first part by showing that mammalian cells, like bacteria and yeast, can carry out homologous recombination, an exchange of matching DNA sequences that had already been demonstrated in bacteria more than fifty years earlier by the 1958 laureate Joshua Lederberg.

Evans solved the second part by finding a cell type, the embryonic stem cell, that could carry a modification into the mouse germ line.

Neither solution alone was enough; the cell types Capecchi and Smithies first worked with could not give rise to reproductive cells, so their modifications could not be inherited until combined with Evans's ES cells.

How does homologous recombination let scientists edit a single gene?

Homologous recombination is a natural process in which two DNA sequences that closely resemble each other exchange pieces of genetic material. It normally happens when chromosome pairs swap sections during the production of egg and sperm cells, which increases genetic variation between generations.

Capecchi and Smithies reasoned that if they introduced into a cell a piece of DNA that matched part of a target gene but also carried a deliberate change, the cell's own recombination machinery might swap the new sequence into the matching place in the chromosome.

Capecchi demonstrated that DNA injected into mammalian cells was indeed incorporated through this route, and that a defective gene could be repaired this way.

Smithies, working on blood diseases, showed the same thing for a human gene and found the targeting worked regardless of whether the gene was active.

A difficulty was that homologous recombination is rare: according to the scientific background document, it occurs roughly a thousand times less often than random insertion of DNA elsewhere in the genome. Capecchi solved this with his positive-negative selection strategy.

1. A targeting DNA fragment is built carrying the desired change, a gene for resistance to the antibiotic neomycin inside the target sequence, and a second marker gene (thymidine kinase) just outside it.
2. The fragment is introduced into cultured cells, where a small fraction undergo correct homologous recombination and most undergo random insertion elsewhere.
3. Growing the cells with neomycin kills cells that never took up the fragment, keeping only those with the resistance gene (positive selection).
4. Treating the surviving cells so that only cells without the thymidine kinase marker survive removes the randomly inserted copies, because random insertion carries that marker along with it, while correct targeting leaves it out.
5. What remains, after both steps, is an enriched population of cells in which the target gene has been changed exactly as intended.

Draw a chromosome with the target gene, a targeting DNA fragment carrying a neomycin-resistance gene inside the matching sequence and a thymidine-kinase gene just outside it, and two arrows showing the two possible outcomes: correct homologous recombination (keeps neomycin resistance, loses thymidine kinase) and random insertion elsewhere (keeps both markers). Label which cells survive each selection step.

How do embryonic stem cells carry a gene change into a whole mouse?

An early mouse embryo, a few days old, is called a blastocyst. Its outer layer goes on to form the placenta, while an inner clump of cells, the inner cell mass, would normally develop into the embryo proper.

Evans found that cells taken from this inner cell mass could be grown in culture without losing their ability to become almost any cell type in the body; he called these embryonic stem cells (ES cells).

Unlike ordinary adult, or "somatic", stem cells, which are each committed to producing only one family of tissue, ES cells are far more flexible: they can develop into any cell type needed to build a new individual.

This property made them the missing "vehicle" that could carry a laboratory-made gene change into a living, breeding animal.

Evans showed that ES cells injected into a blastocyst mix with the host embryo's own cells, producing a chimeric mouse made of cells from two different origins.

When such a mosaic mouse was bred, some of its offspring inherited genes that had come only from the injected ES cells, proving the change could pass down according to ordinary inheritance.

How is a knockout mouse actually made?

Once homologous recombination in cultured cells and ES cells that could reach the germ line were both available, the final step was to combine them into a single working procedure for producing live, gene-targeted mice.

1. Grow mouse ES cells in culture and introduce the targeting DNA fragment, designed to disrupt or change one chosen gene.
2. Use positive-negative selection to identify the rare ES cells in which the fragment has recombined correctly into the target gene.
3. Inject these modified ES cells into a blastocyst taken from a mouse of a different coat colour, so that ES-derived tissue can later be recognised by eye.
4. Implant the injected blastocyst into a foster mother, who carries it to term and gives birth to a chimeric pup made of both the host embryo's cells and the modified ES cells.
5. Breed the chimeric mouse; pups that inherit the modified gene from the ES-cell lineage now carry the change in every cell, including their own germ cells.

Draw, in one row, ES cells in a dish being targeted and selected, then injected into a coloured blastocyst; in the next row, show the blastocyst implanted into a foster mother and the resulting chimeric pup, with an arrow to its own offspring carrying the targeted gene throughout their bodies.

The Nobel committee noted that the first published reports of mice produced this way, using homologous recombination in ES cells, appeared in 1989, and that the number of reported knockout mouse strains rose exponentially afterwards.

The technique can now also be used to switch a gene on, alter its function, or replace it with another version, not only to delete it.

How did the discovery unfold?

Why does gene targeting matter for medicine?

By the time of the award, the press release recorded that more than ten thousand mouse genes, roughly half the genes in the mammalian genome, had already been knocked out, with international efforts aiming to produce knockout versions of every gene.

Gene targeting had, by the time of the award, yielded more than five hundred different mouse models of human disorders, among them cardiovascular and neuro-degenerative diseases, diabetes and cancer.

Capecchi's own later research used gene targeting to uncover genes guiding mammalian organ development and body-plan formation, shedding light on some human inborn malformations.

Evans developed mouse models of the inherited lung and digestive disease cystic fibrosis to study its mechanisms and test gene therapy.

Smithies, besides working on cystic fibrosis, built mouse models of the blood disease thalassaemia and of common diseases such as hypertension and atherosclerosis, using mice engineered to carry one, two or three copies of a blood-pressure gene to study how gene dosage affects disease.

The committee stated that the discovery's "impact on the understanding of gene function and its benefits to mankind will continue to increase over many years to come".

Open questions still include how complex, multi-gene diseases such as hypertension are shaped by interactions between many targeted genes at once, which remains an active area of research rather than a settled picture.

What ethical questions does this work raise?

Because gene targeting relies on breeding and experimenting on live mice, the popular information page notes that the prize "opens up for discussion" whether it is right to use animals for research at all.

It states that every animal experiment in this field is reviewed by an ethical board, which weighs the animals' potential suffering against the expected future benefit to human health.

One argument given in the source is that targeted, accurate mouse models of disease can save human lives, and that better, more reliable models actually mean fewer animals are needed overall to reach useful results, compared with less precise methods.

Mice carrying a human version of a gene, for example a human serotonin receptor linked to depressive disorders, were also described as making drug testing more informative, since the human and rodent forms of such receptors can differ.

These are the committee's own stated considerations rather than a verdict on the ethics of animal research in general, and the source material does not resolve the underlying debate, only describes the review process used to manage it.

How does this connect to what you study?

School biology lessons on DNA, genes and inheritance connect directly to this prize. The idea that chromosomes come in pairs, one from each parent, and that matching DNA sequences can exchange material, is exactly the process of homologous recombination that Capecchi and Smithies exploited.

Lessons on cell division and stem cells also link here: an embryonic stem cell is simply an early, unspecialised cell with the ability to become many different tissue types, the same basic idea taught when comparing embryonic cells with the specialised cells of an adult body.

Understanding why a single gene change needs to reach the germ line (the cells that form eggs or sperm) to be passed on to the next generation is also a direct application of basic Mendelian inheritance, where only changes in reproductive cells are inherited by offspring.

Finally, the mouse models of disease described here, for cystic fibrosis, thalassaemia, hypertension and cancer, are worth remembering as concrete examples of how studying one organism's genes can inform understanding of human disease, a theme that recurs across biology and general-studies questions on biotechnology and genetics.

Quick facts for exams

The Nobel Prize in Physiology or Medicine 2007 was awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies, each receiving one third of the prize, for discovering principles of introducing specific gene changes in mice using embryonic stem cells. The prize was announced on 8 October 2007 by the Nobel Assembly at Karolinska Institutet.

Capecchi and Smithies showed that homologous recombination could target a chosen gene in mammalian cells; Evans isolated embryonic stem cells that could carry such a change into a living, breeding mouse.

Together these discoveries created gene targeting, the basis of "knockout mice" used across modern biomedical research, from basic genetics to disease modelling.

Note: Source. The prize facts in this note are from the Nobel Prize's official site, nobelprize.org.

Glossary

• DNA — the molecule that carries the genetic instructions of an organism, arranged along chromosomes.
• Gene — a specific stretch of DNA that carries the instructions for one trait or function.
• Chromosome — a packaged strand of DNA; in mammals these occur in pairs, one from each parent.
• Homologous recombination — the exchange of matching DNA sequences between two DNA molecules.
• Embryonic stem cell (ES cell) — a cell from an early embryo that can develop into almost any cell type in the body.
• Blastocyst — an early mouse embryo a few days old, with an outer layer and an inner cell mass.
• Gene targeting — the technique of making a precise, intended change to one chosen gene.
• Knockout mouse — a mouse in which a specific gene has been deliberately disabled.
• Chimeric mouse — a mouse made of cells from two different genetic origins, such as a host embryo and injected ES cells.
• Germ line — the cells, such as eggs and sperm, through which genetic material is passed to the next generation.
• Positive-negative selection — Capecchi's method of using two marker genes together to isolate correctly targeted cells.
• Transgenic mouse — a mouse carrying foreign DNA inserted randomly into its genome, rather than at a chosen site.
• Totipotent — able to give rise to every cell type needed to build a whole new organism, including extra-embryonic tissue such as the placenta; ES cells are pluripotent, not totipotent, since they cannot form the placenta.

Common errors and misconceptions

• Misconception: The three laureates each discovered the same thing independently. Correct: Capecchi and Smithies independently discovered homologous recombination in mammalian cells, while Evans independently discovered embryonic stem cells; the prize was shared because both discoveries had to be combined.
• Misconception: Gene targeting and transgenic mice are the same technique. Correct: Transgenic mice carry DNA inserted at a random site, while gene targeting places a change precisely at the matching location of a chosen gene.
• Misconception: A knockout mouse always has its gene completely removed. Correct: Gene targeting can also switch a gene on, alter its function, or replace it, not only disable it.
• Misconception: Any adult cell can be used in place of an embryonic stem cell for this technique. Correct: Adult, or somatic, stem cells are each committed to one line of differentiation, unlike ES cells, which can form almost any cell type.
• Misconception: Homologous recombination is a laboratory invention. Correct: It is a natural process, already shown in bacteria by Joshua Lederberg decades earlier; the laureates learned to exploit it in mammalian cells.
• Misconception: The technique has been used only to study rare, single-gene diseases. Correct: It has also been applied to common, complex diseases such as hypertension, atherosclerosis and cancer.
• Misconception: This prize means the human genome itself was edited. Correct: Gene targeting has been carried out in mice, not in humans.

Exam-style questions with model answers

Q1. State the exact citation for the Nobel Prize in Physiology or Medicine 2007. [2 marks]

1. The prize was awarded "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells".

Q2. Name the three laureates and their share of the 2007 Nobel Prize in Physiology or Medicine. [2 marks]

1. Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies each received one third of the prize.

Q3. Explain the "two-part problem" that gene targeting had to solve. [4 marks]

1. The first part was finding and altering one specific gene among billions of base pairs of DNA without disturbing the rest of the genome.
2. Capecchi and Smithies solved this by showing that homologous recombination, the exchange of matching DNA sequences, works in mammalian cells, letting a targeting DNA fragment swap into the correct place.
3. The second part was ensuring the change reached every cell of a living animal, including its reproductive cells, so it could be inherited.
4. Martin Evans solved this by isolating embryonic stem cells, which could be modified in culture and then carried, through chimeric mice, into the mouse germ line.

Q4. What is an embryonic stem cell, and why was it essential to gene targeting? [4 marks]

1. An embryonic stem cell is a cell taken from the inner cell mass of an early embryo, the blastocyst, which can develop into almost any cell type in the body.
2. Unlike adult somatic stem cells, which are each committed to one line of differentiation, ES cells are far more flexible.
3. Evans showed ES cells injected into a blastocyst could contribute to a chimeric mouse, and that genetic changes carried in the ES cells could be inherited by the chimera's offspring.
4. Without ES cells, Capecchi's and Smithies's gene modifications could be made in cultured cells but could not be passed on to a living, breeding animal.

Q5. Describe Capecchi's positive-negative selection method and explain why it was needed. [5 marks]

1. Homologous recombination occurs far less often than random insertion of DNA elsewhere in the genome, so most cells taking up a targeting fragment have it inserted at the wrong place.
2. Capecchi designed a targeting fragment carrying a neomycin-resistance gene inside the target sequence and a thymidine-kinase gene just outside it.
3. Growing cells with neomycin kills any cell that failed to take up the fragment at all, which is the positive-selection step.
4. A further treatment then kills cells that still carry the thymidine-kinase marker, which happens only when the fragment inserted randomly rather than by correct homologous recombination, which is the negative-selection step.
5. Only cells in which the fragment recombined precisely into the target gene survive both steps, making it practical to find rare, correctly targeted cells among millions.

Q6. Outline, in order, how a knockout mouse is produced once a gene has been targeted in ES cells. [5 marks]

1. Modified ES cells, selected by positive-negative selection, are injected into a blastocyst taken from a mouse of a different coat colour.
2. The injected blastocyst is implanted into a foster mother, who carries it to term.
3. The resulting pup is a chimeric mouse, made up of cells from both the original embryo and the injected ES cells.
4. The chimeric mouse is bred, and pups that inherit the targeted gene from the ES-cell lineage carry the change in every cell of their bodies, including their own germ cells.
5. These pups, and their descendants, are now stable knockout mice that can be studied or bred further.

Q7. Discuss, with examples from the sources, why gene targeting matters for medicine. [6 marks]

1. Gene targeting allowed scientists to disable a single gene and observe the effect in a whole living animal rather than only in cultured cells, which cannot show diseases such as hypertension or diabetes.
2. More than ten thousand mouse genes, roughly half of the mammalian genome, had been knocked out by the time of the award, and over five hundred mouse models of human disorders had been produced.
3. Evans developed mouse models of cystic fibrosis to study disease mechanisms and test gene therapy.
4. Smithies built mouse models of cystic fibrosis, thalassaemia, hypertension and atherosclerosis, including mice with different numbers of copies of a blood-pressure gene.
5. Capecchi's later work used gene targeting to uncover genes controlling mammalian organ development, shedding light on inborn malformations.
6. The committee stated that the discovery's impact on understanding gene function and its benefits to humanity would keep increasing over many years, showing that the significance of the work was expected to grow well beyond 2007.

Q8. What ethical concern did the Nobel committee raise about this prize, and what argument did it give in response? [3 marks]

1. The committee noted that the prize raises the question of whether it is right to create genetically modified mice for research at all, beyond ordinary breeding.
2. It stated that every such animal experiment is reviewed by an ethical board, which weighs the animals' potential suffering against the expected future benefit.
3. One argument given is that more accurate, targeted mouse models can reduce the overall number of animals needed, since their results are more reliable than those from less precise methods.

Key takeaways

• Capecchi, Evans and Smithies shared the 2007 Nobel Prize in Physiology or Medicine for gene-targeting principles using embryonic stem cells.
• Capecchi and Smithies independently showed homologous recombination works in mammalian cells, letting a chosen gene be altered precisely.
• Evans isolated embryonic stem cells, which could carry a gene change into a living mouse and on to its offspring.
• Capecchi's positive-negative selection method made it practical to find rare, correctly targeted cells among many randomly altered ones.
• The first gene-targeted, inheritable knockout mice were reported in 1989, after the earlier 1980s discoveries were combined.
• By 2007, more than ten thousand mouse genes and over five hundred disease models had been produced using gene targeting.
• Gene targeting differs from older transgenic methods, which insert DNA randomly rather than at a chosen site.
• The committee linked the prize to ethical review of animal research, weighing animal suffering against expected medical benefit.

Test yourself

Who were the three laureates of the 2007 Nobel Prize in Physiology or Medicine?

Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies shared the prize equally for gene targeting using embryonic stem cells.

What natural process did Capecchi and Smithies exploit to target genes precisely?

They exploited homologous recombination, the exchange of matching DNA sequences, which lets an introduced DNA fragment swap into the correct chromosomal location.

What did Martin Evans isolate, and why was it important?

Martin J. Evans isolated embryonic stem cells from mouse embryos, which could carry a gene change into a living mouse and pass it to offspring.

What is a blastocyst?

A blastocyst is an early mouse embryo a few days old, with an outer layer forming the placenta and an inner cell mass that can form the embryo.

Why was positive-negative selection needed?

Because homologous recombination happens far less often than random DNA insertion, so a selection method was needed to isolate the rare, correctly targeted cells.

Give one disease studied using gene-targeted mouse models mentioned in the sources.

Cystic fibrosis was studied in mouse models developed by both Evans and Smithies to understand disease mechanisms and test treatments.

How many mouse genes had been knocked out by the time of the award?

More than ten thousand mouse genes, roughly half of the mammalian genome, had already been knocked out by the time of the 2007 award.

What ethical issue did the Nobel committee link to this prize?

The committee linked the prize to the question of whether creating genetically modified mice is ethically justified, noting that animal experiments are reviewed to weigh suffering against benefit.