Functional Genomics and Rat Models

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Insight/Outlook Functional Genomics and Rat Models

Howard J. Jacob The Laboratory for Genetic Research, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226 USA

The 20th Century has seen a remark- lished, most focused on evaluating the breast cancer, cardiovascular diseases, able number of inventions and techno- biology and/or the pathobiology of the cancer, comparative genomics, dental logical advances in virtually all aspects rat. In contrast to its central role in the diseases, diseases of the skin and hair, of human life and health care. Many ar- study of behavior, biochemistry, neuro- endocrinology, eye disorders, growth eas of biomedical research have made biology, physiology, and pharmacology, and reproduction, hematologic disor- great strides in unraveling the cause of the rat has lagged far behind the mouse ders, histology, kidney diseases, meta- human disease and in developing new as a genetic “model” organism, until re- bolic disorders, neurological and neuro- therapies to counter, or at least improve, cently. muscular diseases, nutrition, patho- outcome from disease. However, the Historically, rat genetics had a sur- physiology, pharmacology, pulmonary cause of the vast majority of common prisingly early start. The first genetic diseases, physiology, reproductive disor- disease remains poorly defined. In the studies were carried out by Crampe from ders, skeletal disorders, sleep apnea, final year of the millennium, the release 1877 to 1885 and focused on the inher- transplantation and immunogenetics, of the draft sequence of the human ge- itance of coat color (Lindsey 1979). toxicology, and urological disorders nome promises to bring in a new era for Hugo De Vries, Karl Correns, and Erich (Gill et al. 1989; Greenhouse et al. 1990; basic science research and, hopefully, Tschermak rediscovered Mendel’s laws James and Lindpaintner 1997). unprecedented growth in our under- at the turn of the century, and Bateson standing of human disease. For this to used these concepts in 1903 to demon- Why Rats? occur there is a critical need to annotate strate that rat coat color is a Mendelian the genomic sequence with gene func- trait (Lindsey 1979). The first rat inbred With all the success in the mouse ge- tion and basic biology. Typically, the strain, PA, was established by King in nome project, including the initiation of view from the geneticist immediately 1909—the same year that inbreeding be- the mouse genomic sequencing project turns to mouse, as the mammalian con- gan for the first inbred strain of mouse, and the prospect of having every mouse tributor. Yet, not all biologists are will- DBA (Lindsey 1979). Despite this paral- gene knocked out, why should there be ing to convert to the mouse as their sys- lel start, the mouse soon became the interest in rat? Although there are many tem of choice, in many cases because of model of choice for mammalian geneti- ways to answer this question, the funda- the existence of better models. Although cists, whereas the rat became the model mental reason is the biology. However, I the mouse is undoubtedly going to play of choice for physiologists, nutritionists, think it is important to illustrate how a major role in contributing to the an- and other biomedical researchers. Ge- much research is conducted in rats. notation of gene function, other mam- neticists preferred the mouse because of Most investigators are aware that the rat malian species will also make significant its smaller size, which simplified hous- remains a major model system inside contributions. This Insight/Outlook ing requirements, and the availability of the pharmaceutical industry; yet most piece focuses on the role the rat will play many coat color and other mutants ex- are surprised as to how much research is in annotating the genome in the func- hibiting Mendelian patterns of inherit- conducted in rats inside academia. One tional genomics era. ance, which had been collected by of the major reasons for this surprise is The laboratory rat, Rattus norvegicus, mouse fanciers (Nishioka 1995). Physi- the lack of a vocal rat community. In- was the first mammalian species domes- ologists and other biomedical research- vestigators that use rats tend to be dis- ticated for scientific research, with work ers favored the rat because its larger size ease based and not model based. For ex- dating back to before 1850 (Lindsey facilitated experimental interventions. ample, in my own research, I have 1979). From this auspicious beginning, Over time a large number of rat strains worked in rat, mouse, zebrafish, and hu- the rat has become the most widely were used to develop disease models by man. Inside the National Institutes of studied experimental animal model for selective breeding, which “fixes” natural Health (NIH) extramural program, the biomedical research. Since 1966 (the disease alleles in particular strains or number of research grants funded using earliest year covered by the Medline da- colonies (Greenhouse et al. 1990). For rats on average nears parity for most in- tabase), nearly 500,000 research articles example, there are inbred strains of rats stitutes and centers (Fig. 1). Finally, reporting the use of rats have been pub- used for research in the following areas: comparing a Medline search on rat ver- addiction, aging, anatomy, autoim- sus mouse reveals that rat research is E-MAIL [email protected]; FAX (414) 456-6516. mune diseases, behavior, blood diseases, much more mechanistic in content

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Insight/Outlook

has been an international effort, albeit a relatively small group of 13 labs that I know about (Rat Genome Groups). This international team has now built the be- ginnings of a rat “backbone map” using the rat radiation hybrid (RH) panel. This backbone map contains the vast majority of genetic markers from the major laboratories that developed the markers on a single RH map. Rat is the first organism with a dense, single integrated map, yielding a means to integrate all previously mapped genes and quantita- Figure 1 The relative ratio of grants funded for research using rat and mouse. The results come tive trait loci onto a single backbone. from searching CRISP (Computer Retrieval of Information on Scientific Projects) reports from the NIH This backbone map forms the founda- for fiscal year 1998. For a variety of reasons CRISP reports are not very accurate; so a ratio between tion of the recently U.S. sponsored RGD, mouse grants and rat grants was used. In this way, the inaccuracies of the CRISP reports are hopefully at least consistent. A ratio of 1 means the same number of grants is funded within the institute or which is coordinated with RATMAP, the center. A ratio >1 indicates more rat grants are funded, and a ratio <1 indicates more mouse grants Mouse Genome Database (MGD), and are funded. NHGRI does not show as funding rat. This is an example of the inaccuracies of CRISP, as the National Center for Biotechnology NHGRI has contributed resources to the rat genome project. Abbreviations: National Institute on Information (NCBI). Alcohol Abuse and Alcoholism (NIAAA), National Institute on Aging (NIA), National Institute of Allergy and Infectious Diseases (NIAID), National Institute of Arthritis and Musculoskeletal and Skin Diseases The development of genomic re- (NIAMS), National Cancer Institute (NCI), National Institute of Child Health and Human Development sources for the rat has been enhanced by (NICHD), National Institute on Deafness and Other Communication Disorders (NIDCD), National the U.S. RGPEST project that has just Institute of Dental and Craniofacial Research (NIDCR), National Institute of Diabetes and Digestive been renewed and whose 3-year goals and Kidney Diseases (NIDDK), National Institute on Drug Abuse (NIDA), National Institute of Envi- ronmental Health Sciences (NIEHS), National Eye Institute (NEI), National Institute of General Medical are to identify 60,000+ unique rat genes Sciences (NIGMS), National Heart, Lung, and Blood Institute (NHLBI), National Human Genome (UniGenes) and to map over half of Research Institute (NHGRI), National Institute of Mental Health (NIMH), and National Institute of them using the rat RH panel, as well as Neurological Disorders and Stroke (NINDS), National Center for Research Resources (NCRR). the funding of the RGD. In addition the German Genome Project has just been (data not shown). Therefore, it seems Heart, Lung, and Blood Institute funded for the construction of a physi- logical to position the rat field so the (NHLBI), has funded the Rat Genome cal map for the rat. Notably, the U.S. mechanistic, disease-based research can Project (RGP), the Rat Expressed Se- and German projects continue to coor- be integrated into the awesome power of quence Tag (RGPEST) Project, and the Rat dinate their activities. Consequently, the human and mouse genome projects. Genome Database (RGD) to develop im- the infrastructure for the rat is rapidly Progress of the Rat Genome Project portant genomic tools and resources reaching a level whereby sequencing the that will further enhance the power of genome is the next logical choice. Recognizing the usefulness of the rat as a rat model systems (Table 1). However, it model system, NIH, led by the National is important to point out that this effort To Sequence or Not To Sequence

Given that the mouse genomic sequenc- Table 1. Rat Genome Resources ing project has started with the primary Sponsors purpose to provide a means to annotate the human genome with function. Resource U.S. project international total What is the rationale for sequencing Genetic map >5,000 markers >4,000 ∼10,000 the rat genome project anytime Large mapping cross >1,000 meioses >1,000 soon? The answer is in the comparative genomics that can be conducted ןcoverage ∼20 ןcoverage ∼10 ןYAC libraries ∼10 ∼ ∼ with three mammalian species versus ןcoverage 10 ןPAC library 10 ןcoverage ∼10 ןBAC library ∼10 Low resolution RH panel 106 hybrids 106 hybrids two. First, the rat and the mouse have High resolution RH panel in process? been evolutionary separated for ∼16 mil- ∼ Low resolution RH map >10,000 markers >4,000 markers 11,000 lion years, whereas human and a com- FISH mapping ∼75 genes ? >75 genes Normalized libraries 12 different tissues ? >12 libraries mon rat–mouse ancestor have been split cDNA project >100,000 ? for ∼80 million years. These estimates Rat genome database Rat Genome Database RatMap are derived using the equation r = K / Physical map in process (2T), in which r is the rate of mu- Sequencing the genome ? ? tation, K is the distance, and T is the

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Insight/Outlook divergence time between the two species and the meeting participants generated all genes one at a time or in combina- (Li 1997) and the estimates of K and r the following partial list of priorities tion will replicate all of human biology from Makalowski and Boguski (1998). that need to be fulfilled: in a single species. The baseline biology They also conclude that human and ro- of any species provides strict, although, dent may be too distant to detect subtle 1. Although the production of trans- as of yet, undefined, boundaries that differences in substitution distances genic rats is routine, the creation of cannot be crossed. Consequently, the within murine (Makalowski and Boguski knockout has not been possible, as next millennium is likely to be domi- 1998), whereas rat and mouse appear to embryonic stem (ES) cells have not nated by groups that use the best models have very similar substitution rates. Yet, yet been possible in the rat. There is a and systems to address specific ques- the mouse and rat are very different bio- critical need to develop ES cells or tions. Research groups focused on a logically, despite their similar appear- nuclear transfer (cloning) techniques single species are likely to become ex- ance. Consequently, the similarity com- in the rat. tinct. Therefore, the increase in sequenc- bined with the different biology and a 2. There is a critical need to develop the ing capacity and the continual drop in relatively long evolutionary distance Rat Genetic Resource Center to pre- cost suggest that as many organisms as provides unique comparative opportu- serve and distribute the wealth of rat possible should be sequenced at least at nities. Second, the rat is better character- models to the research community. the cDNA level, but scientists must pre- ized physiologically. Therefore, the op- 3. Strengthening the Rat Model User sent compelling arguments and a cohe- portunity to link the physiology of the Research Community via the Rat sive scientific community to lobby for rat to the mouse via comparative ge- Community Forum Web site (http:// the limited research dollars available for nomics is an advantage to the mouse taiji.ifrc.mcw.edu/RCF), rat-“specific” sequencing. As for the rat, it is a power- community. Third, the ability to link genomic/genetic-based meetings, ful platform for discovery of gene func- the genetic power of the mouse to the and symposia on rats in conjunction tion. The basic tools are in place, and the rat via comparative genomics is a major with mouse and human genetics resources required to “finish” the job are help to the rat community. However, meetings is needed. Upcoming rat relatively minor compared with the in- the most compelling argument is that meetings are to be held at Cold vestment already made in defining the the use of three mammalian species of- Spring Harbor Laboratory, December biology and pathophysiology of this or- fers the opportunity to triangulate ge- 9–12 1999, followed by a meeting in ganism. The rat offers many advantages netics, physiology, and clinical medi- Gothenberg, Sweden, July 2000. for identification of gene function that cine. Sequencing the rat genome in par- 4. Development of a third generation relate to common human diseases, be- allel or immediately following the map consisting of single nucleotide cause of the existing body of knowledge mouse provides the gold standard for polymorphisms (SNPs) would greatly of physiological mechanisms, the avail- comparative genomics and brings the facilitate the positional cloning of ability of models that mimic these dis- nearly 200 years of pharmacological/ disease genes. eases, the ease of breeding, and the abil- toxicological data from the rat and drug 5. A BAC clone resource with an average ity to generate new and better models discovery into genomic context with insert size of 150 kb and 15-fold cov- that match subsets of patients at both human and mouse. erage will be needed for sequencing the phenotypic and genomic levels (Gill the rat genome. et al. 1989; James and Lindpaintner 6. The rat should be positioned for ge- Needs 1997; Jacob 1999). Once genes and their nomic sequencing as soon as pos- functions are identified in rats, patho- Whereas the rat platform is steadily sible. physiologic mechanisms can be eluci- moving forward both in genetically de- Summary dated, and human genetic counterparts fined strains and synthetic strains with can be more easily identified. transgenic technology, the rat system Nearly 10 years ago, when I began build- does require some additional infrastruc- ing genomic tools for the rat as a post- ture. At a recent rat priority meeting doctoral fellow in the laboratory of Eric REFERENCES sponsored by Dr. Harold Varmus and Lander, I was presented with a question: Gill III, T., G. Smith, R. Wissler, and H. the NHLBI, a small group of investiga- Which will occur faster, building the ge- Kunz. 1989. The rat as an experimental tors were asked what additional infra- nomics for the rat or the physiology of animal. Science 245: 269–276. structure was needed for the rat. This the mouse? Now, I have a different view. Greenhouse, D.D., M.F.W. Festing, S. Hasan, 1-day meeting organized by the trans- Given the power of genomics and the and A.L. Cohen. 1990. Inbred strains of NIH Rat Planning Group brought to- strengths of various models, can we af- rats. In Genetic monitoring of inbred strains gether >20 investigators from a broad ford to exclude one model for another in of rats (ed. H.J. Hedrich). Gustav Fischer Verlag, Stugart, Germany. range of disciplines. In addition, a sur- advance of really knowing very much Jacob, H.J. 1999. Physiological genetics: vey was sent out to several thousand in- about gene function? It is critical to re- Application to hypertension research. vestigators to canvas the needs of the alize that no amount of ethylnitrosyl Clin. Exp. Pharmacol. Physiol. community. The results of this survey urea (ENU) or systematic knock out of 26: 530–535.

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Insight/Outlook

James, M. and K. Lindpaintner. 1997. Why mouse and rat genes. J. Mol. Evol. Cambridge, MA, USA; Mark Lathrop, map the rat? Trends Genet. 13: 171–173. 47: 119–121. Michael James, and Tony Monaco, Li, W.-H. 1997. Molecular evolution. Sinauer, Nishioka, Y. 1995. The origin of the Oxford, UK; Norman Lee, Rockville, MD, Sunderland, MA. common laboratory mice. Genome USA; Go¨ran Levan, Go¨teborg, Sweden; Lindsey, J.R. 1979. Historical foundations in 38: 1–7. Klaus Lindpaintner, Boston, MA, USA; the laboratory rat. In The laboratory rat Rat Genome Groups: Pieter DeJong, Buffalo, Tadao Serikawa, Kyoto, Japan; Val (ed. H.J. Baker, J.R. Lindsey, and S.H. NY, USA; Detlev Ganten and Margit Sheffield, M. Bento Soares, and Tom Weisbroth), pp. 1–36. Academic press, Knoblauch, Berlin, Germany; Peter Casavant, Iowa City, IA, USA; Claude New York, NY. Goodfellow and Linda McCarthy, Szpirer, Brussels, Belgium; Takeshi Makalowski, W. and M.S. Boguski. 1998. Cambridge, UK; Howard Jacob, Anne Wantanabe, Tokushima, Japan; Ron Synonymous and nonsynonymous Kwitek-Black, and Peter Tonellato, Wilder and Elaine Remmers, Bethesda, substitution distances are correlated in Milwaukee, WI, USA; Eric Lander, MD, USA.

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Functional Genomics and Rat Models

Howard J. Jacob

Genome Res. 1999 9: 1013-1016 Access the most recent version at doi:10.1101/gr.9.11.1013

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