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Designer Mice Aim Gene Targeting at Mysterious Childhood Cancer
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A laboratory mouse in which a gene affecting hair growth has been knocked out (left), is shown next to a normal lab mouse. (Courtesy of genome.gov)
| Introduction
A Nobel Idea
Knock Outs and New Design
The All Powerful Ones
A Biologic Mystery Begging to Be Solved
Location, Location, Location
Timing Matters, Too
Not Too Early, Not Too Late
A Protective Niche Harbors Dangerous Cells
The Next Moves
Following the Impossible Dream
References and Additional Resources
Glossary
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Introduction
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It’s a long way from Mario Capecchi’s early childhood as an abandoned street urchin in fascist Italy during World War II to becoming a leading scientist of his era. The road linking these two periods was strewn with adversities, but Capecchi saw them as incentives to persevere. He also used great creativity (perhaps inherited from his poet mother) when helping to develop a technique called gene targeting.
Gene targeting makes it possible to create specialized mouse models for studying human biology and disease. It has proven so valuable that it earned Capecchi and two co-developers, Martin J. Evans and Oliver Smithies, the 2007 Nobel Prize for Physiology and Medicine. They developed the basic technique during the 1980s, but they and others continue to refine it and make it more powerful. Capecchi recently used gene therapy to take on a mysterious childhood cancer.
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A Nobel Idea
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Genes were long known to be involved in inherited diseases like cystic fibrosis and sickle cell anemia. It’s increasingly clear, though, that genes also influence – and are influenced by – cancer, obesity, heart disease, diabetes, arthritis, and aging; psychiatric disorders like substance abuse, anxiety, and schizophrenia; and neurodegenerative diseases like Parkinson’s and Alzheimer’s. But what are those genes, and where are they among the tens of thousands of genes on the 23 pairs of human chromosomes? The massive Human Genome Project from 1990 to 2003, which the National Institutes of Health (NIH) helped fund to identify genes, has provided a treasure trove of information for researchers.
However, identifying genes was just the first step, since genes do not come with instruction manuals. How does a gene work in a living body? What happens when it’s mutated? How do mutations cause disease? It’s impossible – and it would be immoral – to do such experiments on people! That’s where gene targeting came to the rescue.
Researchers have used gene targeting to create more than 7,000 designer mice to “model” particular diseases. These animal models are unveiling biological secrets like how the nervous system develops and how our immune system fights off invaders. The mice are leading to discoveries about many human diseases and disorders at a depth never before possible.
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Knock Outs and New Design
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In gene targeting, scientists snip a gene out of a chromosome, and either modify it in the test tube or substitute an entirely different gene. Then, scientists add copies of the modified genes to cells in laboratory cultures, along with enzymes that help insert that gene back into the chromosome.
Beforehand, though, scientists add control codes to the beginning and end of the target gene. For example, stop codes in front essentially “delete” the gene, creating knock out mice. Researchers can infer the missing gene’s normal function by seeing what happens without it. People compare this method to figuring out how a car engine works by popping the hood, taking out one part at a time, and seeing what goes wrong when you turn on the engine.
Scientists also insert new genes or modifications to existing genes and use “start” codes to turn them on in mice. Then scientists can study how genetic variations and mutations affect normal biology or lead to disease. For example, modifying a gene that influences body fat in one way can make a mouse obese on a lean diet. Modifying it another way can keep a mouse lean on a fatty diet.
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The All Powerful Ones
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To produce the actual mice sporting these modified genes, scientists insert the genes into embryonic stem cells that can develop into embryos. They select the cells with properly inserted genes and grow them into embryos. The embryos are implanted in a surrogate mouse mother that gives birth to genetically modified mice. These mice become the models for exploring the function of the inserted gene.
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A Biologic Mystery Begging to Be Solved
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In 2007 Capecchi used a sophisticated version of gene targeting to discover the hidden origin of a rare childhood cancer, synovial sarcoma that currently has no cure or effective treatment.
Sarcomas are cancers that produce tumors in soft tissues. Though rare in adults, they are the most frequent childhood cancers. Synovial sarcoma accounts for 10% of sarcomas, with about 900 new cases a year in mostly adolescents and young adults. Tumors commonly appear near joints of knees, ankles, hips, and shoulders. By the time of diagnosis, the cancer has often spread to the lungs, lymph nodes and bones, and 80% of these patients die.
No existing drugs work well against synovial sarcoma. To design more effective drugs, scientists must know where and when the cancer first arises in the body. In which tissues does it start – muscles or joints? At what developmental stage – before or after birth, during grade school or high school years?
Synovial cancer was a complete biological mystery, except for one telling detail. A fusion gene causes the cancer. Fusion genes happen when a cell mistakenly joins broken chromosomes together in a way that combines two genes from different chromosomes. This fusion, called a translocation, produces a mutant protein.
Scientists had tried using gene targeting to create a mouse model with this fusion gene so they could learn more about the cancer. But the mice always died. The mutation must be lethal, except in specific cells that go on to produce tumors. Capecchi reasoned that if they could activate the mutation in just those specific cells, they could have a mouse that could survive and “model” synovial cancer.
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Location, Location, Location
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Normally, the body knows to turn on a gene only in certain cells and tissues. For example, skin cells turn on a completely different profile of genes than brain cells or kidney cells. Also, many mutations cause harm only in specific organs.
Researchers wanted a way to limit an inserted gene’s activity to just certain tissues in their animal models. During the 1990s, Capecchi and other research teams developed a technique using specialized codes attached to the gene. The codes keep the gene turned off until scientists inject an enzyme, called Cre. This enzyme snips out the codes and allows the mutation to pop out and become active in just a specific tissue. This type of control over gene activity is called conditional expression. (When cells turn on genes, they “express” them.)
Capecchi hypothesized that in synovial sarcoma, the fusion gene is first activated in skeletal muscles because that is where most of the tumors appear in humans. Thus, he used conditional codes that would only turn the gene on in muscle cells.
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Timing Matters, Too
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However, there are different types of muscle cells from early development through adulthood. Embryonic and prenatal stages have progenitor muscle cells. Satellite cells (muscle stem cells) and myoblasts (immature, precursor muscle cells) appear after birth. Mature muscle fibers (myocytes and myofibers) develop later.
Most likely the fusion gene only led to synovial sarcoma when it turned on in one developmental stage. But which one? To find out, Capecchi painstakingly used six different codes to activate the fusion gene in different stages of muscle development.
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Not Too Early, Not Too Late
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Activating the translocation gene in embryonic stages caused the mice to die. Activating it too long after birth allowed the mice to survive without developing cancer.
But turning on the translocation mutation very shortly after birth (in myocytes) led to cancer in these mice 100% of the time. (This stage in mice may correspond to an early childhood stage in humans. That means the cancer might originate more than 10 years before the tumors become evident.)
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A Protective Niche Harbors Dangerous Cells
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The synovial sarcoma tumors in mice looked and acted the same as human tumors do, even at the molecular level – a hallmark of a good animal model. For example, the tumors in mice usually appear at the joints of limbs, just as they do in people. Studying the new mouse model showed why.
When Capecchi activated the mutation, most cells could not survive – so they couldn’t go on to produce tumors. Joints, however, secrete a protective factor that keeps the nearby cells alive even after activating the mutation. Those survivors go on to develop additional mutations that promote cancer. That’s why most tumors appear near joints.
Learning more about that protective factor, and how to counteract it, may provide clues for fighting the cancer. But the animal model just became available in 2007, and it’s a long road from there to having a new drug approved for human patients.
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The Next Moves
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Scientists everywhere may now use this mouse model of synovial sarcoma to explore important questions. What is the second thing that happens after the translocation gene is activated? Must other mutations happen before the muscle cells become malignant? Can we develop a “smart drug” that targets the translocation to cure or prevent this cancer? Scientists are optimistic, because smart drugs that target other fusion genes in other cancers have worked well.
Also, scientists can adapt this gene targeting technique to develop animal models of other diseases that have defied scientific understanding, including psychological disorders and mental illness. For example, Capecchi created a mouse that models a form of obsessive compulsive disorder! The possibilities, he has said, are limited only by the imagination of the researcher.
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Following the Impossible Dream
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Gene targeting is now so commonplace that researchers do not even footnote it anymore in their papers. But back in 1970s, it seemed impossible to study human genes in mice.
Then, Capecchi observed that mammalian cells seemed to “know” how to take in a foreign gene. He realized that cells must have some machinery, or enzymes, that he could exploit to control the process. So he asked the NIH for a grant to work on inserting human genes into mouse chromosomes. The NIH thought the idea could never work and denied the grant. But Capecchi privately pursued his hunch. A year later he sent a revised proposal to the NIH, which they accepted. In time, this NIH-supported research led to an entirely new, widely used way to study biology – and to a Nobel Prize in Physiology and Medicine for Capecchi.
What seemed impossible when he began his career is now among the most productive and promising areas of biological research. The lesson Capecchi draws from both his personal history and the development of gene therapy is the same: Anything is possible.
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References and Additional Resources
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References
- Learn.Genetics, Genetic Science Learning Center. (2007). How to build a knockout mouse. Retrieved from http://learn.genetics.utah.edu/features/knockout/index.cfm\
- National Human Genome Research Institute. (2007). Knockout mice. Retrieved from http://www.genome.gov/12514551
- Learn.Genetics, Genetic Science Learning Center. (2007). Capecchi wins 2007 Nobel prize in physiology or medicine. Retrieved from http://learn.genetics.utah.edu/features/capecchi/index.cfm
- Haldar, M., Hancock J. D., Coffin C. M., Lessnick S. L., & Capecchi M. R. (2007). A conditional mouse model of synovial sarcoma: insights into a myogenic origin. Cancer Cell, 11(4), 375-88.
- Davis, S. R., & Meltzer P. S. (2007). Modeling synovial sarcoma: Timing is everything. Cancer Cell, 11(4), 305-7 and 375-88.
- Capecchi, M. R. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nature Review Genetics. 6(6), 507-12.
- National Institutes for Health, NIH News. (2007). NIH grantees win 2007 nobel prize in physiology or medicine for developing techniques to target specific genes in mice. Retrieved from http://www.nih.gov/news/pr/oct2007/od-08.htm.
- Boguski, M.S. (2002). The mouse that roared. Retrieved from http://www.nature.com/nature/journal/v420/n6915/pdf/420515a.pdf.
- Nature. (2002). Initial sequencing and comparative analysis of the mouse genome. Retrieved from http://www.nature.com/nature/journal/v420/n6915/full/nature01262.html.
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Additional Resources
NIH Web Sites
Researcher's Web Site
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