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P&S Journal

P&S Journal: Winter 1997, Vol.17, No.1
Coming Soon To a Lab Near You: A Mouse Model For Breast Cancer

Knockout mice are
transforming the way
researchers study disease

M.D./Ph.D. trainee Stephen Tsang with a chimeric mouse

By Devera Pine

W e're expecting...and the bundle of joy--if all goes well--will be the first viable "knockout" mouse for the human breast cancer gene BRCA1. It's a delivery that could change the fate of women with breast cancer while adding to the arsenal of new research weapons developed in the medical field.

Sometime within the next year or so, scientists--perhaps at P&S--will announce the creation of viable knockout mice for BRCA1. Since the event will mark a major advance in creating an authentic model for breast cancer, researchers around the country are working furiously to be the first to announce such an arrival.

Targeted Disruption of Pdeg Locus

Knockout Mice: A Sampling Of P&Amp;S Research

P&S and other labs already have produced mice that harbor non-working copies of the BRCA1 gene. But, in addition to its functions in breast tissue, BRCA1 is important to the development of the mouse embryo--so important, in fact, that mice with non-functional copies of the gene die before they are born.

Knockout and transgenic mice provide key new tools for understanding human biology. "It used to be, the most powerful systems for developmental biology were frogs--which had big embryos that could be manipulated--or fruit flies, on which we could do rapid genetic studies," says Dr. Franklin D. Costantini, professor of genetics and development. "But now mice have become the most powerful system for many types of developmental studies."

What are knockout and transgenic mice, and what do they mean to the future of medicine?

Fancy Mice

In the 1980s, researchers began developing the two technologies needed to make knockout mice a reality: embryonic stem cells (ES cells) and homologous recombination to achieve gene targeting. Two labs, in Cambridge, England, and in San Francisco, developed embryonic stem cell technology at about the same time. Techniques for gene targeting in mammalian cells were first developed at the University of Wisconsin, the University of Illinois at Chicago, and the University of Utah. At P&S, Drs. Pamela L. Schwartzberg (a 1992 P&S graduate now at the National Institutes of Health), Elizabeth J. Robertson (now at Harvard), and Stephen P. Goff collaborated to combine these two techniques, producing the first gene-targeted mice.

Embryonic stem cells are undifferentiated cells from the early stage of a mouse embryo and have the ability to develop into any type of cell. To begin the process of making knockout mice, scientists first modify a stretch of DNA so that the gene they are studying is disrupted, or knocked out. They then exchange this modified DNA for the endogenous DNA sequence in the stem cell--a genetic bait and switch ploy.

Scientists have several methods of ensuring that this genetic sleight of hand "takes." For instance, along with the disrupted gene, the modified DNA also will encode resistance to a specific antibiotic. Once the modified DNA has been inserted into the stem cells, the cells are bathed in antibiotics. This kills all stem cells that do not have the antibiotic resistance gene, thus eliminating any cells where the DNA integration didn't work.

The modified stem cells are next injected into a "foster" blastocyst, which is then implanted into the womb of a "foster mother" mouse. The trick here is that the original stem cells come from a mouse of one color, such as brown, and the foster blastocyst comes from a mouse of a different color--say, black. This is done so that each mouse born has visible evidence that it carries the modified DNA: If the "brown" stem cells successfully become part of the black foster blastocyst, the resulting mouse will have a mixed brown and black coat. This mouse is called a chimera--after the mythological Greek beast with the head of a lion, body of a goat, and tail of a serpent.

The chimeric mouse carries copies of both the modified and unmodified ("wild type") DNA. With any luck, the ES cells contribute to the mouse's germ line (sperm cells, since the ES cells are always XY and therefore only male mice are used), so that it can pass the knockout mutation on to its progeny. To that end, the chimeric mouse is mated with a black mouse, producing some offspring that have at least one copy of the modified DNA. Those offspring are then mated to each other, producing some mice with two copies of the modified gene--knockout mice.

A different process is used to make a transgenic mouse, in which a gene (human, mouse, or otherwise) is micro-injected into a one-cell mouse egg and incorporated into a chromosome. This process does not replace a mouse gene (as in a knockout mouse); instead, the new gene co-exists with the original mouse genes, over-expressing a gene the mouse did not have before.

Disease Models

The ability to manipulate genes this way has far-reaching consequences. First, knockouts go far beyond the genetic techniques of Mendel: Instead of starting with a phenotype and working backward to determine the genotype, genes are manipulated so scientists can see the resulting phenotype firsthand. "We can now ask what happens to an animal if you eliminate a gene," says Dr. Stephen P. Goff, the Higgins Professor of Biochemistry and a Howard Hughes Medical Institute investigator. Reverse genetics--making a mutation in a gene and then determining the phenotype--has been "unbelievably important" the past 20 years in understanding gene function. "But only in the past seven years have we been able to do it in a whole mammal," he says.

In a whole animal, knockout techniques can reveal the normal function of a "disease" gene: A cancer gene often also has a role in the normal development of an embryo, for instance. This is a logical connection, since both cancer and the development of an embryo involve rapid cell growth, says Dr. Virginia Papaioannou, professor of genetics and development.

Knockout mice also can serve as disease "models." To mimic a genetic predisposition to breast cancer in humans, researchers will disrupt only one of the two copies of the BRCA1 gene (one copy, or allele, from each parent) in a mouse. Scientists can then use this model to develop theories about why breast cancer develops and to test treatments, especially genetic therapies to correct the mutation.

In addition to providing clues to diseases such as Alzheimer's, Huntington's, and leukemia, mice can answer questions about mood, for example. Dr. René Hen, associate professor of pharmacology in the Center for Neurobiology and Behavior, is studying approximately 15 receptors for serotonin in the brain. Scientists believe that serotonin has a role in obsessive-compulsive disorder, aggression, anxiety, appetite disorders, and depression. But because serotonin is found all over the brain, it is impossible to determine which neural circuits are important to which disorder.

By knocking out the genes that encode the various receptors, Dr. Hen is slowly connecting the receptors to the moods they help control. For instance, when he knocked out the 5-HT1B receptor, the resulting mice were more likely to react in novel environments. This hyper reactivity is a trait found in attention deficit disorder. In addition, the mice were more aggressive, more sexually active, and more impulsive.

Knockout mice reacted more severely when exposed to cocaine and consumed more alcohol than normal mice. Dr. Hen emphasizes that this does not mean he has found the gene for drug abuse or aggression: The genetic components are only part of the factors that account for these disorders. But, he says, "This gives us a model for what might happen in humans with a deletion or mutation in that particular receptor." Dr. Hen is now investigating the genetics of humans with alcohol or aggression problems to see if they carry a mutation in the 5-HT1B receptor.

"We Have a Dead Mouse"

Most of the time, development of knockout mice is anything but straightforward. "If you start with a cloned gene and everything works perfectly, you will have a knockout mouse in nine months to a year," says Dr. Papaioannou. "But that's only if everything works the first time, which never happens in science."

Germline transmission of mutant allele

Instead, researchers generally encounter a few bumps along the way. For instance, genes usually have multiple effects throughout life, turning on and off at various points as needed. But if a researcher knocks out a gene that is needed during the development of the embryo, the mouse may never make it past the embryo stage. "That's when I get called in," says Dr. Papaioannou. "They say, 'We have a dead mouse. What do we do?'"

The discovery that a gene is essential for early development is an important part of understanding human biology, Dr. Papaioannou says. For instance, the failure of the BRCA1 mice to develop reveals a previously unsuspected role for a gene that is also involved in neoplasia. The result may provide important clues about the underlying common role for this gene product in different circumstances.

When a gene is essential to early development but scientists want to study its function in later life, they turn to another technique, known as a conditional knockout. In a sense, conditional knockouts are genetic time bombs: The DNA is engineered so the target gene is knocked out at a specific time in the mouse's lifespan. That way, if the gene is essential to development of the embryo, it can still function during that stage. Later, when the mouse is fully grown, the gene can be turned off. Conditional knockouts have another advantage: The DNA can be engineered so the target gene is knocked out only in a specific body part.

The BRCA1 gene is a good example of this: Because the gene is essential to embryo development, researchers now want to knock it out when the mouse is an adult. Since the gene is involved in breast cancer, the logical place to knock out the gene is in the breasts.

To make a conditional BRCA1 knockout, researchers create a mouse in which the BRCA1 gene is flanked by DNA repeats of 34 base pairs, known as "loxP" sites. Next, researchers create a mouse that has a breast-specific promoter (a DNA sequence that "turns on" a gene, in this case, only in the breast). The breast-specific promoter is linked to sequences coding for an enzyme--called cre--that hunts for and cuts at loxP sites. The researchers then cross the two mice, creating a mouse with the loxP sites, BRCA1, cre, and the breast-specific promoter. When that mouse grows up, the breast-specific promoter turns on, starting a domino effect: The promoter activates the cre gene, which, in turn, hunts for the loxP sites and, finding them, cuts at the sites, deleting any sequences in between. The end result is an adult mouse in which BRCA1 has been knocked out in the breast tissue.

Many researchers around the world are following this kind of strategy in the race to make the first conditional BRCA1 knockout. But since the technique of making conditional knockouts is new--only a few labs have been successful so far--the process is slow. At P&S, for instance, Dr. Thomas Ludwig, assistant professor of anatomy and cell biology, believes he is about one year away from having a BRCA1 mouse.

The chimeric mice in this litter have spotted coats.

Another mouse model that calls for a conditional knockout is the mouse for Huntington's disease. Dr. Scott Zeitlin, assistant professor of pathology, and colleagues developed a regular knockout for Huntington's, but the embryo did not survive. The protein the gene coded for is essential for embryonic survival and may be involved in programmed cell death or apoptosis. With the gene knocked out, apoptosis increased so much that the embryos died at a very early stage.

Dr. Zeitlin and colleagues are now working on a conditional knockout. Dr. Zeitlin has an additional goal, however--to make a "knock-in" mouse, which would include the repeating pattern of bases that constitute the genetic defect of Huntington's. This model would allow researchers to test drugs and to examine aspects of the disease that cannot be studied in people. For instance, researchers can look for presymptomatic changes in the brains of knock-in mice. They also could vary the length of the repeating pattern of bases, to see how that changes the severity of the disease. This is an important process; generally, the longer the repeat region, the more severe the disease. The knock-in mouse also could help determine if other factors, such as another gene, are involved in the disease. Dr. Zeitlin estimates that both a conditional knockout and a knock-in mouse are six months away.

Rodents of Tomorrow

In the meantime, researchers continue to develop and study other varieties of knockout mice. Dr. Goff, for instance, studies the c-abl gene, the first autosomal gene ever knocked out (by Drs. Goff, Schwartzberg, and Robertson, in a study published in 1989). The gene affects many different systems, including the development of B cells and how cells respond to interleukins.

Other knockout mice have led to a better understanding of retinitis pigmentosa (a genetic disease that causes blindness), increased knowledge of the genes involved in the development of an embryo, and insights into diseases ranging from Alzheimer's to sickle cell anemia.

The technology involved in knockout mice goes hand in hand with the Genome Project, says Dr. Costantini. "With the Genome Project, people have been pulling out thousands of new genes and not really knowing what they do. Knockouts enable them to find out. Knockouts have revolutionized developmental biology and mammalian genetics."

Courtesy of Stephen Tsang, M.D.'98/Ph.D.'96

Targeted Disruption

Embryonic stem cells are isolated from embryos of a mouse strain with a specific coat color, such as brown. DNA is manipulated to create the desired mutation. The cells are then put through a selection step to ensure that only cells with manipulated DNA are retained. The manipulated stem cells are injected into the blastocyst of a mouse with a different coat color, such as black. The blastocyst is then implanted in a foster mother and allowed to develop into mice. Any mice that contain the manipulated DNA will have a multicolored coat of brown and black and are known as chimeras. If the sperm in the mice also derive from the genetically manipulated cells, the mice will pass the mutation through the germ line and are known as germline chimeras. The sperm of the chimeras contain one allele that has the knocked out DNA sequence and one "normal" allele. The chimeras are crossed with a mouse with two normal alleles. Of the resulting offspring, some will have one normal and one manipulated allele. Two such mice are then crossed to produce a knockout mouse, in which both alleles are manipulated.

Knockout Mice: A Sampling Of P&Amp;S Research

Gene Related Human Disease(s) Laboratory
cRet (tyrosine kinase) Potter syndrome
Hirschsprung disease
Dr. Franklin Costantini
Il6 Osteoporosis Dr. Franklin Costantini
Epor Benign familial erythrocytosis Dr. Franklin Costantini
Hd Huntington's disease Drs. Argiris Efstratiadis and Scott Zeitlin
Brca1, Brca2 Familial breast cancer 1 and 2 Drs. Argiris Efstratiadis and Thomas Ludwig
Ghr (Growth hormone receptor) Laron dwarfism Dr. Argiris Efstratiadis
cAbl Chronic myelogenous leukemia Dr. Stephen Goff
Pdeg (Phosphodiesterase gamma) Retinitis pigmentosa and macular degeneration Dr. Stephen Goff (in collaboration with Dr. Peter Gouras)
Htr1b Aggression Dr. René Hen
Bcl6 B-cell lymphoma Dr. Riccardo Dalla-Favera
Various odorant receptor genes Anosmia to specific odorant Dr. Richard Axel
Pka Age-related memory loss Dr. Eric Kandel
Chart courtesy of Stephen Tsang, M.D.'98/Ph.D.'96 / Chart design by Howard R. Roberts

copyright ©, Columbia-Presbyterian Medical Center

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