Both emerging and well-established animal models are providing researchers novel insights into human biology. Diana Gitig explores new genetic approaches being used in the rat to uncover the basis of human disease and finds out just what the naked mole rat could tell us about how we age.
Laboratory rats are a traditional animal model, used by scientists for over a hundred years. Pathologists, physiologists, neuroscientists, and certainly behavioral and cognitive researchers prefer them to mice because their physiology and circulatory systems are more akin to ours—not to mention, they are easier to manipulate because of their larger size. Moreover, detoxifying enzymes in the rat are similar to ours in both number and type, so they can provide more relevant results in phamacodynamic and toxicology studies. But one hurdle has prevented their use in genetic studies: the dearth of functional rat embryonic stem (ES) cells has made it difficult to create transgenic rats.
Sangamo BioSciences Inc., in Northern California, has developed a technology for creating transgenics that circumvents the need for ES cells. The company's approach relies on the use of zinc finger nucleases (ZFNs). Zinc fingers are DNA binding domains found naturally in transcription factors that specifically recognize three nucleotides. Sangamo has created a library of zinc fingers that can be linked together to create specificity for a longer DNA sequence. Then, any given combination can be grafted onto a restriction endonuclease that will cut at that predefined sequence. A left and right ZFN—one to cut each strand of DNA—are injected into the nucleus of a one-celled embryo. The generated double-strand break induces the cell's nonhomologous end-joining DNA repair machinery, which is prone to error, resulting in mutations that yield truncated or non-functional protein products. Using ZFNs, a gene knockout can be made in a matter of months.
According to Elizabeth J. Wolffe, director of corporate communications at Sangamo, each zinc finger had to go through an iterative process to optimize specificity. “The big breakthrough was automating the technology. Now, you can feed your sequence into a database and it will give you the six best combinations,” says Wolffe. Aron Guerts, an assistant professor of physiology at the Medical College of Wisconsin (MCW) who was first author on the article that first described rat knockouts using ZNFs (1), agrees. “Sangamo has worked very hard on the technology to keep improving the molecules and building libraries of zinc finger proteins which can be combined to make highly active ZFNs,” he says. “This was the key to getting it work efficiently enough to try in an embryo.”
In July 2007, Sigma-Aldrich licensed the technology from Sangamo. Sigma calls their molecules CompoZr ZFNs and uses the molecules at their Sigma Advanced Genetic Engineering (SAGE) Labs, which recently obtained a grant from the Michael J. Fox Foundation to develop models for Parkinson's disease. While mouse models of Parkinson's disease have been made in the past, pheno-typically the mice don't shake. Edward Weinstein, director of SAGE, said that the ZFN-generated knockout rats “provide a huge opportunity for moving science and drug development further, and for improving quality of life.” Dave Smoller, president of Sigma Research Biotechnology, can barely contain his enthusiasm. “We need to find models that really show efficiency,” he says. “The cost for proving that drugs work is so prohibitive; if we can save time, we can save money and spare people pain.”
Guerts and Howard Jacob, his colleague at MCW, have used ZFNs to knock out rab38, which has been implicated in end-stage renal disease in the fawn hooded hypertensive rat (2). Raymond Buelow, founder and CEO of Open Monoclonal Technology (OMT), Inc. and the senior author of the Science paper announcing the rat knockout model, has now used ZFNs to knock the IgM gene out of rats in order to make human monoclonal antibodies. Buelow does note, however, that genetic manipulation of the rat using ZFNs is still a work in progress. “These are knockout rats, not transgenic rats”, he says. “We have not yet shown that zinc finger nucleases can stimulate homologous recombination, which would be nice because then we could make knockins with targeted integration.” But researchers are hoping to apply this technology to that goal soon.
So far, ZFNs have been used to generate knockouts in fruit flies, worms, zebrafish, and cultured cells. Their use in rabbits has recently been reported, and knockout crickets, mice, and salamanders are also in the works. “In the next few years,” says Guerst, “you will see all kinds of amazing genomes being manipulated by ZFNs.”One for the ages
Rats, like mice, have short life spans, generally living 2–5 years. This is convenient for longitudinal studies, but hardly appropriate for studying aging in humans. “The whole aging field has been focused on short-lived species,” notes Vera Gorbunova, an associate professor of biology at the University of Rochester whose research is focused on aging, DNA repair, and cancer. “To understand human [aging], we must use a model that is more long-lived.”
Enter the newest—or depending on how you look at it, the oldest—rodent model organism: the naked mole rat. Naked mole rats are the longest living rodents, with recorded life spans of over 28 years in captivity. Native to East Africa, naked mole rats live in large colonies in underground burrows. They are also capable of breeding for their entire lives. While for most species, maximum life span is usually correlated with body size, naked mole rats—like humans—are outliers with longevity quotients far exceeding that expected for their size (approximately 35 g). Add to this their phylogenic relationship to mice and rats, along with the fact that the species doesn't appear to get cancer, and several researchers suspect naked mole rats could prove beneficial in comparative aging studies.
Aging can be defined as a gradual decline in fertility and cellular and organ functioning that eventually results in—well, death. There have been a number of theories presented to explain the mechanisms of how and why animals age. One of these is telomere maintenance. Telomeres are the ends of chromosomes that cannot be replicated by normal means. Human germ cells express telomerase to maintain telomere length, but human somatic cells do not. As a result, telomeres get shorter with each ensuing cell division until their reduced length signals the cell to enter replicative senescence. In contrast, mouse somatic cells express telomerase and do not senesce in culture. It had been thought that telomere shortening was an anti-cancer safeguard used by large, long-lived animals. But Gorbunova demonstrated that somatic cells in the naked mole rat, like mouse cells, still have high telomerase activity. Telomerase activity, then, appears negatively correlated with body mass, not with lifespan (3).
Oxidative stress is another popular theory of aging. It posits that aging is caused by the accumulated damage of reactive oxygen species (ROS), a byproduct of aerobic metabolism. This theory is particularly appealing because ROS are endogenously and continuously produced, and irreversibly damage DNA and proteins. Yet Rochelle Buffenstein, a professor of physiology at the Barshop Institute for Longevity and Aging Studies at the University of Texas Health Science Center, has shown that even naked mole rat cells taken from young animals have higher levels of oxidative damage than similarly aged mouse cells. The ability to withstand oxidative damage, rather than the amount of damage done over time, may therefore be the more important determinant of longevity.
With these two theories of aging now being questioned, Gorbunova plans to determine if DNA repair is essential to long lifespan like everyone assumes it is. The naked mole rat's curious ability to remain cancer-free may provide some insight.
“Naked mole rats use several strategies for cancer suppression,” Gorbukova suspects. Since 90% of mice get tumors within 2–3 years—and the naked mole rat's cancer incidence is so dramatically opposite—she thinks the difference isn't attributable to one mechanism alone. Although naked mole rat cells do not display replicative senescence because they express telomerase, they do replicate relatively slowly in culture. Gorbunova found that this was due to an alternate strategy to control proliferation, which she deemed “early contact inhibition.” Generally, cell division slows as cell density increases, but naked mole rat cells stop growing at a much lower cell density than both human and mouse cells. “Maybe we can see how these alternative cancer avoidance strategies can be applied to humans,” she says.
Gorbunova's findings, along with Buffenstein's on telomerase, ROS, and early contact inhibition, have begun to uncover the mechanisms responsible for the 10-fold difference in life span between the naked mole rat and its more common lab rat relative. Future studies will greatly aid in understanding the various molecular processes underlying aging. “Perhaps naked mole rats may use some strategies for life span extension that humans don't use,” says Gorbunova, “or that humans do use but [which] hasn't been explored in depth.”