Test Tubes With Tails

The relationship between man and mouse has had, at times, a strained history: They were vilified in the Book of Leviticus; their most feared enemy, the cat, was deified in ancient Egypt; and their English name evolved from the derogatory Sanskrit mush, meaning "to steal."1 Over the centuries, a more amicable rapport grew between Asian and European breeders and their furry pets, prized for their exotic color and behavior. Yet only a few scientists prior to 1900 took advantage of the creatures' ub

Feb 4, 2002
Brendan Maher
The relationship between man and mouse has had, at times, a strained history: They were vilified in the Book of Leviticus; their most feared enemy, the cat, was deified in ancient Egypt; and their English name evolved from the derogatory Sanskrit mush, meaning "to steal."1 Over the centuries, a more amicable rapport grew between Asian and European breeders and their furry pets, prized for their exotic color and behavior. Yet only a few scientists prior to 1900 took advantage of the creatures' ubiquitous presence.

But, as the laws of Gregor Mendel were being rediscovered, the mouse began nosing its way into the scientific sphere. Before 1900, mice had been used for comparative anatomy and air quality experiments, but later, scientists chose them to test Mendel's theories because of their small size, quick breeding, and easily discernable variation. The mouse was on the way to helping reinvent biological science—and become reinvented as well.

Tens of millions of mice are bred each year to, among other things, mimic human diseases and screen the effects of drugs and toxins. Ninety percent of research mammals are mice, which are now the focus of the second biggest public genome project. The mouse has marked its place in basic research, even more in the past two decades, as new techniques emerged to genetically manipulate them to model diseases more precisely. But it's still a little early to get from mouse to cure, says Kathleen Murray, director of transgenic services at Charles River Laboratories, the world's largest supplier of these test tubes with tails.

Early History Makers: A Breed Apart

A farm in Granby, Mass. is an ancestral epicenter for hundreds of strains used today. Retired schoolteacher Abbie Lathrop bred mice that were derived from stocks traded among Chinese, Japanese, and European fanciers. These "fancy" rodents, prized for their exotic fur colors, caught the attention of Harvard biologist William Castle. In 1901, he saw the potential in using these easily discernable variations to test Mendel's theories. Though similar research was happening in France and England, Castle's work garnered him the title of great-grandfather of mammalian genetics.1 As genetic variants dwindled, however, so did some researchers' interest. The pot needed stirring, and in 1909, the ladle holder was Harvard undergrad Clarence Cook Little.
Courtesy of Charles River Laboratories

C.C. Little's DBA (for dilute, brown, and non-agouti) was the first inbred mouse.



Wanting to transplant and study tumors in mice, Little recognized the need for a more homogenous breed. He inbred brothers and sisters past 20 generations to create animals that would not reject tumor grafts. Despite criticism from people like Castle, who felt that the compromised health of consanguineous couplings would reduce their value, and others who morally opposed forced incest, Little went ahead.2 His end result, DBA, was bred to express recessive characteristics—dilute, brown, and non-agouti. The first inbred strain, DBA is hailed by many as the birth of the modern lab mouse. Explains Donald W. Bailey, a senior staff scientist emeritus at Jackson Laboratory (Jax), which Little founded, "It surreptitiously came in very useful, but very quickly there were other strains like C57 Black [C57BL]."

Up and into the 1920's, the number of inbred strains grew with additions like Halsey Bagg's Bagg albino (BALB/c), Little's C57BL and C57BR, and Leonell Strong's first tumor-prone mouse strain, "A." These and their substrains forged a permanent place in scientific literature; their lineage lives on, often for reasons that were unfathomable at the time. For example, in 1928, geneticist Leslie C. Dunn developed strain 129, which has a high incidence of testicular cancer. This line supplied the first embryonic stem cells, making possible many modern genetically manipulated mice.

In 1915, J.B.S. Haldane began linkage studies, and, by 1935, rudimentary linkage maps had been created for nearly a dozen genes, such as pink eye, dilution and short ear. Up until this time, inbred mice had only been available to Jax researchers. To eke out a living during the Great Depression, the lab began selling its mice. Within 10 years, the number of mapped genes had doubled. By 1954, the number had grown to more than 70 genes on 20 chromosomes.1

Providing a significant tool for that leap was George Snell, who joined Jax in 1935. Picking up on Little's early work on tumor-transplant resistance, Snell, in the 1940s, developed the first congenic strains, heterozygous at only one locus. Snell mixed two inbred strains and repeatedly backcrossed subsequent offspring with the inbred parental line, while selecting for a desired trait not seen on the parental strain. Snell's work on histocompatibility mechanisms won him the 1980 Nobel Prize in Physiology or Medicine, and congenic strains spurred an explosion in linkage studies. Many call it the most significant event since DBA. "The work of Snell ... to produce inbred animals with different histocompatibility antigens has been maybe the single biggest advance in mouse research because it's really allowed all kinds of things to be done," says Jon Gordon, professor of Ob/Gyn and Mathers Professor of geriatrics, The Mount Sinai Medical Center in New York.

The Atomic Age: Accidents Will Happen

In the Cold War era, the US Department of Energy began studying how low-dose radiation affected mammalian genomes. "The main concern was fallout from nuclear tests and things of that sort," says Liane Russell, head of the mammalian genetics and development section, Oak Ridge National Laboratory in Tennessee. Irradiation caused large deletions and translocations, leading to advances in fine-structure mapping of the mouse genome, and other discoveries.

In the 1970s, scientists turned to chemical mutagenesis, having nearly exhausted the uses of radiation. Though many chemicals were tried, ethylnitrosourea (ENU) emerged a winner in 1979. Using the specific locus method, William L. Russell showed that this powerful and still popular mutagen creates single-point mutations around the genome.3 Mutagenesis projects have taken off in the UK, Germany, USA, and Australia, which catalog and provide thousands of new mutants each year.

To many, randomly induced mouse mutations have yet to prove their mettle as a research tool. Jax senior staff scientist Eva Eicher notes that while ENU can produce a wealth of new mutations at a given allele, characterizing those mutations is at least as hard as distinguishing spontaneous mutations that arise naturally. And, sometimes nature's experiments are even better.

Courtesy of Jackson Laboratory

Unfurred: At left, a pigmented nude (B6.Cg-Foxn1nu) mouse; at right, the strain from which it was derived (C57BL/6J) or "Black 6" the most widely used inbred strain.


In 1962, viral epidemiologist Norman Grist came across a spontaneous, hairless, mutant mouse at the Virus Laboratory, Ruchill Hospital, Glasgow. He sent it to the Institute of Animal Genetics in Edinburgh for a genetic workup where the responsible gene was dubbed "nude."4 The nude mouse was subsequently found to lack a thymus, and had no T- or B-cells. Its immunodeficient status soon made it a candidate for human tumor xenografts, allowing scientists to study human tumors and tissues in non-human systems for the first time. "Certainly for the field of organ transplants and immune function, this is an important advance," says Dabney Johnson, a group leader at ORNL.

Courtesy of Charles River Laboratories

SCID Mouse



Adding to the long list of mice that have helped dissect the machinations of the immune system was the discovery of SCID mice (for severe combined immunodeficiency) in 1983 by Melvin Bosma, at Fox Chase Cancer Center in Philadelphia.5 Bosma, now a senior member of the center's basic science division, was typing mouse serums for immunoglobulin variability. His group could not find the allotype of interest in a particular mouse, and subsequently realized that it had no immunoglobulin, period.

SCID mice have seen much use in cancer and immunology research. "[SCID] will continue to be important, because the AIDS problem might be resolved there," says Douglas Coleman, senior scientist emeritus at Jax. Concomitant with Bosma's discovery, new technologies emerged that may even eclipse the vast utility of SCID. "There were a lot of folks that took advantage of [SCID], but it was perhaps superceded by some of the technology that allowed you to do such things by design," Bosma says.

Designer Mice

The late 1970s and early 1980s saw a terrific rush to bring the new and emerging molecular biology and recombinant DNA technologies into the mouse. A seminal moment came in 1980 when Jon Gordon and Frank Ruddle at Yale University created the first transgenic mouse through pronuclear-injection of a fertilized egg cell.6

"At the time," says Gordon, "we were not at all concerned with issues like making a mouse with thalassemia or sickle cell anemia. We just wanted to know, 'Could an animal that had been through 30 million centuries of evolution accept DNA [to which] it had no biological relation and still develop?'" Researchers didn't even check for expression of the inserted gene, a thymidine kinase gene from a human herpes simplex virus, though Gordon says he thinks they would have found it. A year later, Gordon and Ruddle, along with other labs, demonstrated germ-line transmission of the foreign gene, and an explosion of transgenic technologies and techniques ensued.

In 1981, geneticist Martin Evans and colleague Matt Kaufman, both at Cambridge University at the time, created the first murine pluripotent embryonic stem cell line. This enabled easier transgenic methods, leading to the first knockout mice by molecular biologist Mario Capecchi, University of Utah, and a Howard Hughes Medical Institute investigator, and geneticist Oliver Smithies, excellence professor, pathology department, University of North Carolina, an achievement for which they and Evans won the 2001 Lasker Award for Biomedical Research.7

Knockouts allow researchers to silence genes by placing foreign sequences within critical exons via homologous recombination. These knockouts and refinements on the techniques continue to be a boon for biomedical research, both in mimicking disease and uncovering gene function. Models now exist that mimic human diseases, ranging from Alzheimer's to cystic fibrosis to heart disease.

With the emergence of more exquisitely targeted mice and the availability of the mouse and human genomes, science is moving into a more prolific arena of research in which key differences between mouse and human systems will be quickly evaluated. But, as with bullets, no magic mice exist, except in Walt Disney's imagination. "There isn't a single genetically manipulated mouse that has been used yet to produce a drug that cures a disease," says Murray of Charles River. "It will probably be a combination of multiple [mice] that all have data that complete the picture."

Brendan A. Maher can be contacted at bmaher@the-scientist.com.
References
1. The Mouse in Biomedical Research Vol. I History, Genetics, and Wild Mice, H.L. Foster, J.D. Small, J.G. Fox, eds., Academic Press, Inc: San Diego, 1981.

2. D.W. Bailey, "Sizing up the set of H Genes in Mice," In: Minor Histocompatibility Antigens: From Laboratory to the Clinic, D. Roopenian, E. Simpson, eds., Landes Bioscience: Georgetown, Texas, 1999.

3. W.L. Russell et al., "Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse," Proceedings of the National Academy of Sciences, 76:5818-9, 1979.

4. S.P. Flanagan, "Nude, a new hairless gene with pleiotropic effects in mouse," Genetical Research, 8:295-309, 1966.

5. M.J. Bosma et al., "A severe combined immunodeficiency mutation in the mouse," Nature, 301:527-30, 1983.

6. "Landmarks: Genetic transformation of mouse embryos by microinjection of purified DNA," The Journal of NIH Research, 6:65-73, 1994.

7. B.A. Maher, "Lasker ceremony: Homage amidst angst," The Scientist, 15[20]:10, Oct. 15, 2001.



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