December 01, 2011

 

 Changing the world with an embryo

 

PennVet researcher awarded National Medal of Science

 
 
posted November 16, 2011

 

Throughout the years, various breakthroughs in experimental modification of the genome have had a substantial impact on civilization.

The domestication of plants and animals about 10,000 years ago, the introduction of selective breeding, and the discovery by Gregor Mendel in the mid-19th century that specific genetic elements could be identified were three developments that ultimately allowed a relatively small percentage of the population to feed the remainder. This freed up of much of the populace to do the other things that account for modern civilization

The fourth and most recent major breakthrough—inserting new genes into the germ line of a developing organism—came in 1981 from the laboratory of a veterinarian, Dr. Ralph L. Brinster. The discovery has allowed researchers to produce animals with selected traits that are indispensable models in understanding life processes and diseases.

For his pioneering work in the field of genetics, Dr. Brinster, the Richard King Mellon Professor of Reproductive Physiology at the University of Pennsylvania School of Veterinary Medicine, has been awarded the 2010 National Medal of Science.

"Dr. Ralph Brinster is a trailblazer in the field of reproductive biology and genetics whose work has had inestimable influence in science and medicine," Penn President Amy Gutmann said in a press release. "His early findings helped usher in the era of transgenic research and represent foundational aspects of techniques used in genetic engineering, in vitro fertilization, and cloning. We are extraordinarily proud that he has received the National Medal of Science in recognition of more than five decades of scientific achievement."

Dr. Brinster, now and then

 Dr. Ralph L. Brinster in 1961 and today

Groundbreaking work

Dr. Brinster's interest in animal genetics and the mammalian germ line dates back to his upbringing on a Cedar Grove, N.J., farm where his family raised dairy goats. He studied animal science as an undergraduate at the Rutgers University School of Agriculture and graduated in 1953. Immediately after, he enlisted in the Air Force, serving for about three years during and after the Korean War. Once he returned from military service, he earned his VMD degree from the University of Pennsylvania School of Veterinary Medicine in 1960.

 

"No one anticipated in the beginning how dramatic the impact would be of modifying, selectively and experimentally, genes and individual DNA sequences in an animal. It has truly revolutionized biology, medicine, and agriculture."

 Dr. Ralph L. Brinster, Richard King Mellon Professor of Reproductive Physiology, University of Pennsylvania School of Veterinary Medicine

 

 

 

 

 

 

 

Dr. Brinster received an AVMA fellowship his first year out of veterinary school that allowed him to pursue postgraduate training at UPenn.

"I didn't have any magic plan or big plan. I just followed my interest and didn't know much about graduate training, but the faculty pointed me in the right direction," he said.

He recalls visiting an Anatomy Department faculty member who told him he didn't belong in that department after Dr. Brinster talked about his interests and, instead, directed him to the Physiology Department, where Dr. Brinster was eventually accepted, earning his doctorate in 1964.

There, he learned how to be an effective researcher.

"I thought it was important to have a system that allowed you to assess if something was having a good effect, bad effect, or no effect," Dr. Brinster said.

"I approach things systematically. I set an objective, and then I determine what kind of resources I have. I develop a plan and work relentlessly. Those are the four steps to achieving anything, and you don't want to get them out of order."

He says it took him three to four years to develop his approach.

He adds that the fifth, and perhaps most important, factor is luck.

"If you can choose between talent and luck, choose luck. I didn't have a choice. I was lucky," Dr. Brinster said.

Whether it was luck, talent, or both, Dr. Brinster started his career with much success when, for his doctoral thesis, he developed the first reliable in vitro culture system for early mammalian embryos. Today, this technique continues to form the foundation for research on mammalian embryos, including technologies such as transgenic engineering, embryonic stem cell therapy, human in vitro fertilization, mammalian cloning, and knockout engineering.

Using this method of embryo manipulation, he next worked out many aspects of the metabolism and development of eggs and early embryos. From there, Dr. Brinster became interested in modifying the development of animals and their germ lines, and he went on to become the first person to show that it was possible to colonize a mouse blastocyst with stem cells from older embryos. Moreover, Dr. Brinster first demonstrated that teratocarcinoma cells could combine with blastocyst cells to form adult chimeric mice, establishing the feasibility of this approach to change the genetic character of mice.

New era in science

In the late '70s, Dr. Brinster became interested in developing methods of introducing nucleic acids directly into an egg. He requested some samples of messenger RNA from the laboratory of Richard D. Palmiter, PhD, a professor of biochemistry at the University of Washington School of Medicine, to inject into mouse eggs in his pilot experiments.

 

Dr. Brinster receives National Medal of Honor from President Obama

Courtesy of the National Science & Technology Medals Foundation

A much-deserved honor

The National Medal of Honor is the highest honor given by the United States government to scientists and engineers. Nominees are selected by a committee of presidential appointees on the basis of their extraordinary knowledge in, and contributions to, chemistry, engineering, computing, mathematics, or the biological, social, and physical sciences.

Dr. Ralph L. Brinster is the first veterinarian to receive the medal since it was created in 1959. He is one of the few scientists from an agricultural background to win the medal, joining the ranks of Nobel Prize laureates Norman Borlaug, an agronomist and humanitarian who has been called "the father of the Green Revolution," and Barbara McClintock, a scientist and one of the world's most distinguished cytogeneticists.

This year, seven eminent researchers were awarded the medal, including Dr. Brinster, during an Oct. 21 ceremony at the White House.

 

 

 

After experiencing some success doing this, Dr. Brinster needed more mRNA, and again turned to Dr. Palmiter for more samples. Later, however, Dr. Brinster began to wonder whether he could inject genes instead of mRNA into the eggs.

"Early on, it wasn't obvious how we could measure the consequences of the genes we proposed, but then, we made the fusion gene and (discovered the) enzymatic function of this gene was easy to assay," Dr. Palmiter said.

Dr. Palmiter had been interested in the regulatory regions of genes, so he sent Dr. Brinster a certain fusion gene. Fast-forward a few months later—it's now 1981—and this fusion gene is injected into fertilized mouse eggs that are then transplanted into pseudopregnant mice. The pups are born, and, sure enough, some of those that had developed from eggs injected with the fusion gene made the specific protein in their liver. The researchers' experiment showed, for the first time, that new genes could be introduced into the mammalian genome. 

Great discoveries

The two researchers hadn't met until November 1981, right before their first paper was published (Cell 1981;27:223-31). They talked about what to do next and settled on using this gene transfer technique to correct a genetic disease. 

This pursuit led to the famous "giant mouse experiment," in which the rat growth hormone gene was expressed in the liver of mice (Philos Trans R Soc Lond B Biol Sci 1984;307:309-12).

"It now seems simple, but at the time, there were lots of known genetic diseases, but the genes involved in general were not known," Dr. Palmiter said.

Together, they, along with their collaborators, developed many of the first models of human disease throughout the 1980s and produced the first transgenic rabbits, sheep, and pigs.

Drs. Brinster and Palmiter also provided the first proof of expression of transgenes, the first example of cancer arising from a transgene, and proof of the targeted integration of DNA by egg injection.

lab mice

 Mice from Dr. Ralph L. Brinster's famous giant mouse experiment, in which the rat growth hormone gene was expressed in the liver of mice and which represented the first published example of transgenesis. "It catalyzed interest in science in the general public. It made people think about how powerful this could be and, in fact, was. So now, thousands of transgenic mice are made every year (for) drug testing and disease (studies), and the way transgenics are made has become very refined to the exact spot where you want the DNA changed. But the first thing was to show you could get in. That was a big breakthrough," Dr. Brinster said.
Courtesy of Dr. Ralph L. Brinster
 

 In an autobiographical essay, Dr. Brinster wrote: "It was an exciting time in science for me, and the field developed rapidly. No one anticipated in the beginning how dramatic the impact would be of modifying, selectively and experimentally, genes and individual DNA sequences in an animal. It has truly revolutionized biology, medicine, and agriculture. Many have since contributed to this transformation in our understanding and ability to modify the genetics of a living organism."

The researchers talked every Saturday after their success introducing genes into the mice. The transgenic mice were made in Dr. Brinster's laboratory in Philadelphia, and tail biopsy samples were sent to Dr. Palmiter in Washington state for DNA analysis.

"We got about 200 tail samples a week and would go through and say which is positive and which is not. ... If we didn't finish Saturday afternoon, then we'd pick up again on Sunday. We did that for almost 15 years," Dr. Palmiter said about their laboratories' various experiments. "It was exciting doing something new and different every year, but in the end ... you get identified with a process rather than biological questions."

Dr. Palmiter went on to pursue his interests in neurobiology in the early '90s, and Dr. Brinster studied male germ cell development. In all, the two have published more than 140 papers together.

Practical applications

To refocus his efforts on male germ cell development, Dr. Brinster needed a system of analysis similar to the one he had developed for eggs in the 1960s that would enable him to assess any experimental effects on spermatogonial stem cells, the male germ cell line stem cells. He hypothesized that if one were to take cells from a fertile testis and put them into the testis of an infertile mouse, the stem cells would be able to develop in the seminiferous tubules and generate donor-derived spermatogenesis. Dr. Brinster was proved right, and over the next few years, his laboratory determined many of the characteristics of spermatogonial stem cells, including their ability to be cryopreserved, which makes individual males biologically immortal. He and his colleagues also demonstrated that these stem cells could survive for months in vitro, which eventually led to culture techniques and the ability to modify the germ line with spermatogonial stem cells. Recently, Dr. Brinster's laboratory has extended their culture methods to farm animals and humans.

One of his lines of investigation is the development of an approach to cryopreserve testicular cells obtained by biopsy from prepubescent boys receiving treatment for cancer that will make them infertile. Following recovery from cancer, the boys could use the cryopreserved testicular cells to re-establish their own spermatogenesis.

"I've always been interested in practical applications (of research). Infertility in animals and livestock is a major problem. A 5 to 10 percent difference in infertility of farm animals can be the difference between success and failure. I just gradually became more interested in the developmental and genetic aspects, but I never lost interest in veterinary application(s)," Dr. Brinster said.

Humility in the midst of success

Receiving the National Medal of Science, Dr. Brinster said, was a surprise and a great honor.

"The medal comes to me, but the really big thing is it recognizes an important event in science. That's what the medal is about. Not me. I'm going to be gone, but the fact people consider this an important breakthrough is what is substantive," Dr. Brinster said about developing the technique of transgenesis.

He says most of his time spent in the laboratory actually involves routine experiments, but he knows that without them, the field will not move forward and he won't get published or receive more funding. These experiments allow him to do the "more interesting things that might change the field."

He owes much of his success to the hard work and talent of his students, colleagues, and collaborators.

"I keep things going and direct them," he said. "They're all smarter than I am."

He applies the same humility to what he says he is most proud of: his wife, Elaine. Together, they have four children. One is a veterinary pathologist, two are surgeons, and another is a trial lawyer.

"I think they mostly have my wife's genes, because they're mostly successful. She has the brains," he said.