Danielle R Reed - US grants

DESCRIPTION (adapted from applicant's abstract): Some humans perceive the taste of phenylthiocarbamide (PTC) and its chemical relative, propylthiouracil (PROP) as intensely bitter at low concentrations, while others find it undetectable, even at much higher concentrations. Although this human trait has been recognized for a long time, and its Mendelian pattern of inheritance documented, the corresponding gene is unknown. Preliminary analysis of 100 families conducted by the P.I. suggests that the gene may reside on the short arm of chromosome 5. Therefore, the P.I. proposes to conduct experiments to precisely map and identify the gene. She plans to test the linkage between chromosome 5p and taster status in an additional 100 newly collected families (Specific Aim 1). This would provide a replicate sample to verify the initial map location, and the pooled sample of 200 families will be sufficiently powerful to meet the genome-criteria of significant linkage. In Specific Aim 1, the P.I. proposes to densely genotype these 200 families using markers in the 5p13 region, construct haplotypes, and find those haplotypes shared among tasters, but not shared among nontasters. The linked area of 5p contains 26 simple sequence repeats and single nucleotide polymorphisms within a 13MB region, which will be used for this dense mapping. In Specific Aim 3, the P.I. will initiate an evaluation of candidate genes located within the region identified, by screening for variabilities associated with PROP-taster status.

Genetic variation in body weight and fatness in mice and humans is best explained by multigenic inheritance. Characterizing genetic involvement in the development of human obesity has been difficult due to lack of environmental and experimental control. Mouse models have proved to be a useful tool in the discovery of genes that influence body size because experimental control can be maintained over breeding and environment. The goal of this project is to identify genes and their alleles in mice that lead to variability in body weight, body length, and adipose depot weight, by tracking the co-segregation of genotypes with these phenotypes in an F2 intercross. Specific Aim l: Genome screen for markers that co-segregate with body weight, body length and adipose depot weight in an F2 intercross derived from the 129/) (129) and C57BL/6ByJ (B6) mouse strains: Hybrid mice from the 129 and B6 strains, phenotyped for body weight, body length and adipose depot weight, will be genotyped using polymorphic loci, and linkage analysis will be conducted to find regions influencing variation in these phenotypes. These strains were chosen for analysis because the 129 strain is lighter and leaner than the B6 strain, and large phenotypic variation exists within the F2 generation. Preliminary results from mouse chromosome 4 gave a LOD score of 8.8 near the leptin receptor gene (Lepr) for body weight, which accounts for approximately 15 percent of the genotypic variance. Specific Aim 2: Saturation genotyping of areas that give preliminary evidence for linkage. When areas of linkage are identified, the flanking regions will be densely genotyped to identify the 1-5 cM region most likely to contain the gene of interest. Specific Aim 3: Evaluation of candidate genes: identification of sequence variation and allele characterization: Candidate genes from the 129 and B6 strain will be sequenced to identify allelic variation that affects body weight, length and adipose depot weight. Mapping of chromosome 4 suggests previously unidentified alleles of Lepr may exist that lead to variation in body weight, body length and adipose depot weight in mice and therefore Lepr will be sequenced using DNA from B6 and 129 mice. Chromosome 2 also appears to contain a gene or genes that give evidence for linkage to body weight (LOD=4.8) and body length (LOD=3.6), and this region contains several candidate genes. The goal of this project is to understand how allelic variability affects body weight, body length, and adipose depot weight in the mouse. Orthologs of these genes may be involved in human obesity, and may have a significant impact on human health.

Orthologs of mouse obesity genes may be involved in human obesity, and have a significant impact on human health. The focus of this application is to identify a gene or genes on mouse chromosome 9 that influence adiposity. As a prelude to this work, we have conducted a genome scan using an F2 population derived from the B6 and 129 strains. A major finding from this genome scan was evidence for linkage of body weight (LOD score--3.8) and adiposity on chromosome 9 (LOD scores=3.2). The locus on chromosome 9 accounts for between 10 and 20% of the total trait variance, and has an additive mode of inheritance. There have been several studies, in addition to this genome scan, that have identified mouse chromosome 9 as an important area of linkage for obesity, yet no fine mapping of this region has been done. This goal will be met by the completion of four Specific Aims: Specific Aim 1: Fine mapping of chromosome 9 using 457 mice f_om the F2 intercross between B6 and 129 strains. A dense map will be created to refine the linkage peak. Specific Aim 2 will be the creation of congenic and subcongenic lines of mice with the plus adiposity allele introgressed into the donor genetic background. Comparison of overlapping donor fimgments will further refine the location of the trait locus, and restrict the number of candidate genes. Specific Aim 3 will be an evaluation of candidate genes: first, genes that are polymorphic between the B6 and 129 strain will be identified by comparison of their sequences (Mouse Genome Sequencing Project and Celera). Then phenotype-genotype correlations among other inbred strains of mice will be conducted, followed by laboratory and in silico assays of the tissue distr_ution of gene expression, and gene expression differences between the B6 and 129 strains for selected tissues. The goal of Specific Aim 4 is to determine gene function of high-priority candidates using transgenic mouse construction, and subsequent phenotype analysis. Understanding the role of each molecule important in obesity will be a significant step towards the development of safe and effective therapeutics. 'ERFORMANCE

In mice, many genomic regions contain variation that results in differences in adiposity (Reed 2003; 2006; 2007; 2008). The goal of this research program is to find the gene or genes on mouse chromosome 9 that account for the quantitative trait locus Adip5, which is associated with increased weight of the gonadal adipose depot. Although several lines of evidence suggest that a gene or genetic variant here has the ability to regulate adiposity, the exact gene or DNA sequence that causes this effect is not known. The QTL Adip5 has features that make it a practical target for a positional cloning approach: it is not particularly susceptible to maternal effects or epistatic interactions, and it is associated with a distinct phenotype (gonadal depot weight). Furthermore, the experimental plan is designed to identify Adip5 if the locus is imprinted (i.e., if there are parent-of-origin effects). Within the current Adip5 confidence interval, there are several credible candidate genes (Bbs4, Cpy19a1, Crabp1, Cplx3, Il18, Lipc, Nedd4), as well as dozens of genes and noncoding RNA of unknown function. Using a chromosome 9 substitution strain developed in our laboratory for this purpose (CSS-9), we will backcross these mice to the host strain (C57BL/6ByJ; B6) and conduct a genome scan to reduce the confidence interval of Adip5 (CSS-9 X B6 N2 genome scan; Aim 1). Based on the refined confidence interval provided by the genetic mapping information, we will parse this chromosome into small intervals through successive breeding cycles, and create microcongenic strains (<200 kb), one of which will contain the gene responsible for Adip5 (Aim 2). To identify the exact gene responsible for Adip5, we will genetically engineer one or more mouse strains with a segment of 129 DNA substituted into a B6 background by homologous recombination, and evaluate its effect on gonadal depot weight (Specific Aim 3). The long-range goal of this work is to develop an approach to systematically identify genes that contribute to normal variation in fatness among mice.

DESCRIPTION (provided by applicant): In biomedical research using animal models such as mice, it is often useful and sometimes essential to understand the animal's metabolic status in different experimental states, for example, before and after drug treatment or as a result of genetic manipulation, selective breeding, or learning. In that spirit, this application focuses on the need of 10 National Institutes of Health-funded projects by seven investigators from the Monell Chemical Senses Center to obtain equipment designed to automatically measure food and water intake, activity, oxygen consumption and carbon dioxide production. The advantage of this system is that it measures key aspects of metabolism of rodents continuously and accurately, which aids in the interpretation and implementation of a variety of studies. No such equipment is currently available within the Monell Chemical Senses Center, an independent, nonprofit research institution. The projects that would benefit from an automated and accurate monitoring of these phenotypes in mice (and other models) relate to the genetics of obesity;the effects of early husbandry on adult phenotypes;alcohol preference behavior among selectively bred strains of mice;the interaction of taste and obesity, food intake, and body weight, especially in mice that are "sweet-likers";treatments specifically targeted at changing dietary composition and fat metabolism;the neural concomitants of nausea and learned taste aversions;the intake and appetite for minerals such as calcium;the effect of inflammation on taste;and genetically engineered mice with compromised or enhanced taste function. This equipment is needed by a large core group of investigators and will enhance the quality of the work done here. Currently, food and water intake data are collected manually, and there are no facilities for the measurement of activity or gas exchange. Translational research in these areas is of critical concern because of the focus on obesity prevention and other diseases of nutrient intake, e.g., alcoholism, osteoporosis, excess sweet intake, and diabetes. PUBLIC HEALTH RELEVANCE: The pattern of food and water intake, activity, and metabolism in mice changes as a result of genetic manipulation or other experimental treatments. The accurate and detailed measurement of these traits in experimental animals enhances the quality and reliability of experimental results. To that end, we are requesting funds to purchase a system that will automatically and continuously measure food and water intakes, activity, and energy metabolism of rodents used as models of human disease.

DESCRIPTION (provided by applicant): Many types of research require accurate measures of body composition. In this application, we are requesting funding to purchase equipment designed to measure body composition (fat, muscle, and water). This equipment will meet the needs of thirteen National Institutes of Health- funded projects conducted by eight investigators at the Monell Chemical Senses Center. Currently, body composition is measured at Monell by chemical analysis, which is slow, or by DEXA, which is limited to mice and requires anesthetization;or we make use of nearby equipment that necessitates sacrifice of animals after measurements are taken. The advantages of the system requested in this application are that (1) it measures composition in tissue samples or in awake animals of several species (by use of animal tubes that restrict movement), (2) it is faster and more accurate than the other methods, and (3) it can measure the animals while they are onsite, without need to sacrifice them afterward. The projects that would benefit from measuring body composition involve the genetics of obesity in mice and rats;alcohol preference among selectively bred strains of mice;the interaction of taste and obesity, food intake, and body weight, especially in mice that are "sweet-liker's";the appetite for minerals such as calcium;the effect of inflammation on taste;and genetically engineered mice with compromised or enhanced taste function or with deficits in gut nutrient sensing. This requested equipment will enhance the quality of the work done by a large core group of investigators. Translational research in these areas is of critical concern because of the focus on obesity prevention and other diseases of nutrient intake, e.g., alcoholism, excess sweet intake, and diabetes. Public Health Relevance: The body composition of animals can differ because of naturally-occurring genetic variation, genetic manipulation, or other treatments, e.g., a diet high in fat or calories. The fast and accurate measurement of body composition in animals enhances the quality and reliability of experimental results. To that end, we are requesting funds to purchase a Bruker minispec LF110 system that will measure the amount of fat, muscle, and water in animal models of human disease.

The primary expertise of Monell investigators spans a wide range of disciplines, including human sensory physiology and psychophysics (Drs. Breslin, Cowart, Dalton, Lundstrom, Pelchat, Mennella, Wysocki. Yamazaki. and Zhao), mouse and/or rat genefics (Drs. Bachmanov, Reed, and Tordoff), molecular biology (Drs. Brand, Margolskee, and Huang), and animal chemosensory physiology (Drs. Beauchamp, Lowe, Margolskee, and Reisert). Most or all of these research programs use DNA or RNA analysis methods at various fimes, but these methods often lie outside their area of expertise. This Core will serve to remove barriers from the invesfigators' use of these techniques. For instance, transgenic mice used by laboratories that study mouse behavior and physiology need to be genotyped to ensure that they are of the correct group (e.g., knockout or wild-type), but the invesfigator has no training in genotyping methods. Therefore, it is useful for the investigator to have a technician available who has been rigorously trained by experts to perform this work, to make use ofthe equipment available in the Core. Thus, not all people who study genefically engineered mice need to have all the equipment to genotype mice in their own laboratories. Likewise, invesfigators doing human sensory work might like to know their subject's genotype for key sensory polymorphisms (e.g., is the person homozygous for certain alleles associated with bitter taste blindness?). Again, this is often outside the expertise of their laboratory, but by working with this Core they can receive appropriate training and readily collect these data. Thus, this Core will provide a centralized common resource and reservoir of experience in genotyping and DNA/RNA analysis that would be inefficient to replicate in each individual laboratory. The availability of Core services extends the capabilifies of the participafing laboratories beyond what each could do individually.

DESCRIPTION (provided by applicant): Despite the widely recognized health impacts of excess body fat in our population, the determinants of individual differences in obesity are not well understood. Studies in mice have led to many breakthroughs in understanding human genetics, including leptin and its receptor, sweet taste receptors, and members of the growth hormone pathway. One source of body size variation we have recently identified is a locus or cluster of loci on mouse chromosome 2 near 160 Mb. We have verified in several crosses that genetic variants in this region relate to large differences in fat mass in mice, and these results are (1) consistent in their direction and magnitude of allelic effect and (2) resilient to changes n environment. To prepare to identify these loci, we used marker-assisted selection to breed two reciprocal chromosome 2 substitution strains that can be used as parents for backcross studies to fine-map the relevant loci (Aim 1). These strains are also a ready-made start point to construct congenics to isolate this region into successively smaller intervals (Aim 2). Once the interval is very small, knockout mice will be measured for fat weight to determine which gene(s) is responsible for these traits (Aim 3). The strategy of Aim 3 makes use of the increased number of knockout mice available as a part of the Knockout Mouse Project (KOMP). The payoff of this project will be to identify a source of natural variation that regulates fat weight in animals. Because many medical problems are due directly or indirectly to changes in body composition, understanding their molecular pathways may point to new avenues to improve human health.

Project Abstract: The Monell Chemical Senses Center Institutional (T32) Postdoctoral Training Program provides a unique opportunity for postdoctoral fellows to obtain training at the world?s foremost center for research into the chemical senses?taste and smell. Twenty-three faculty members have diverse scientific and international backgrounds and conduct collaborative, multidisciplinary research using specialized methods derived from disciplines such as electrophysiology, molecular biology, genetics, analytical chemistry, and psychophysics. Many applicants to the training program have little background in chemosensation but are given the opportunity to apply skills learned elsewhere to chemical senses research. In addition to specialized hands-on research training provided by faculty mentors, fellows attend instructional courses, workshops, and journal clubs; they have opportunities for small-group review of data, one-on-one assistance with clear scientific communication, and instruction on academic ethics tailored to their stage of professional development. A three-person mentoring committee helps formulate an individual training plan for each fellow?s unique career path and then tracks the fellow?s progress, helps set practical scientific goals, suggests skill development opportunities (e.g., learning new statistical or wet-lab techniques), and provides written feedback every six months. There is emphasis on learning the skills to prepare results for publication and to write successful research grants. During the previous funding period, Monell?s T32 program supported nine postdoctoral fellows; four are still in training, and five now have positions in academic institutions or elsewhere in the biomedical research workforce. All the trainees have published papers in appropriate peer-reviewed journals, attended national and/or international conferences, sought specialized training in courses and workshops, and become familiar with new standards for the responsible conduct of research (including rigor and reproducibility). Four trainees have won awards for excellence in research; others have won competitive research grants, including three Individual Research Training Grants (F32s) and one similar governmental award. The long-term goal of Monell?s Postdoctoral Training Program is to train independent scientists in taste and smell biology, part of NIH?s mission to improve the nation?s health.

? DESCRIPTION (provided by applicant): Many types of research require accurate methods of assessing the sequence of genomic DNA and measures of gene expression. In this application, we are requesting funding to purchase a QuantStudio 12K Flex all-in-one qPCR system, which is designed for high-throughput targeted genotyping and for measuring expression of specific genes. This equipment will meet the needs of projects for 17 National Institutes of Health-funded R01 grants held by 12 investigators at the Monell Chemical Senses Center, a nonprofit research facility focusing on chemosensory biology--taste and smell--and its role in nutrition and health, including effects on such diseases as diabetes and obesity. Currently, targeted genotyping is conducted at Monell using an older instrument that can run only a few samples at a time and uses a large volume of reagents, which makes this work both slow and expensive. The advantages of the system requested in this application are that it (1) measures a larger number of samples in less time, (2) needs smaller amounts of expensive reagents to obtain the same information, and (3) offers multiple array configurations, such as Taqman assays and the OpenArray system. This requested equipment will enhance the quality of the work done by a large core group of investigators. The projects that would benefit from fast, high-throughput targeted genotyping involve chemosensory biology and its relationship to health. Translational research in these areas is of critical concern because of the focus on obesity prevention and other diseases of nutrient intake, such as excess sweet intake and diabetes. Chemosensory receptors act in other tissues of the body beyond the mouth and nose where they also affect human health.

? DESCRIPTION (provided by applicant): The aim of this application is to improve the Animal Facility of the Monell Chemical Senses Center and facilitate its accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). A not-for-profit research institute [501(c)(3)], Monell was founded in 1968 with a unique mission to investigate the basic biology of taste and smell. This institute, originally part of the University of Pennsylvania, is in the heart of the University City Science Center, the largest and oldest urban research center in the United States. Monell's research is supported predominantly by the National Institutes of Health and other governmental agencies, such as the U.S. Department of Agriculture. The annual research conducted at Monell totals $16M. Animals -- mostly mice, rats, and ferrets -- are now used by 12 research programs at Monell and account for nearly $6M research dollars spent annually. Most research now is conducted with mice, but in the past, animal studies have used nonhuman primates, meadow voles, musk shrews, mini-swine, and wild-caught animals housed in the facility. Research projects on taste and smell conducted with animals housed at Monell have resulted in discoveries that have influenced the direction and scope of research programs on taste and smell, nationally and internationally. For instance, the sweet taste receptor was identified in a mouse study at Monell, as was the role of the major histocompatibility complex in parents' ability to identify offspring by smell. Monell is also well known for its work on comparative biology. Monell's current facility of animal care was established in 1971. In 1989 it was gutted and rebuilt, in part with NIH funds. The animal care and use program at Monell is well developed: It has operated successfully for the past 43 years and is licensed by the USDA, with no non-compliant findings. Our goal is to update our ~10,800 square foot Animal Facility and obtain accreditation by AAALAC. To accomplish this goal, we need to replace the air-handling unit that supplies the Animal Facility. This unit functions poorly and is in imminent danger of failing due to its age. Its lack of redundancy makes maintenance difficult, because emergency procedures must often be used to support the animal environment while work is performed on it. The prospect of catastrophic failure grows more likely with each passing month. Although temperature and humidity can usually be maintained within the range required for animal safety, these parameters often deviate too much for optimal research conditions, with unacceptable swings in humidity becoming increasingly common. Moreover, the amount of heat recovery has decreased over time and this deficit has accelerated recently due to deterioration of the system's coils. This problem is very difficult to fix, because the tight location of key parts of the unit makes cleaning and servicing the heat exchange coils almost impossible. There is also an immediate concern that leakage in the discharge side of the exhaust ductwork may lead to air-borne contaminants (particulate material) that could affect the health of our animals and interfere with sensory testing in our research programs. Our plan is to replace the aging primary air-handling system with a new system that features redundancy, ease of maintenance access, significantly improved energy recovery, and corrections to the exhaust system to reduce air-borne particulates. This new system is projected to be at least 20% more energy efficient over the existing unit's peak efficiency (which is no longer realized) and will provide significantly better control over temperature and humidity. Monell has a clear line of authority for managing its Animal Facility. Monell's Director supervises the Director of Facilities Management, who manages the Animal Facility and the program of animal care under the guidance of the Institutional Animal Care and Use Committee (IACUC). She has nearly three decades of experience in this role, has managed several major renovations at Monell over the years, and will be the Project Manager. An expert Building Engineer, who is responsible for electrical, plumbing, and mechanical aspects of the buildings, reports to the Director of Facilitie Management. The Principal Investigator of this application is a scientist with 30 years of experience at Monell, with funded projects that rely heavily on mouse breeding and over 100 published papers on animal models of human disease, who also has served on Monell's IACUC. An experienced American College of Laboratory Animal Medicine board-certified laboratory animal Attending Veterinarian has been guiding Monell's program of animal care and use since 1982, including the original design and construction of the Animal Facility, IACUC responsibilities and functions, animal care operations, veterinary care, training, and compliance. The expert Mechanical Engineer that Monell contracted for this application has project management expertise, has participated in many major renovations of this building, and provided detailed line drawings for this application. He complements the expert and experienced team that Monell has assembled for this essential renovation project. Air-handling renovations described in this proposal will result in necessary and important improvements to Monell's facilities of animal care, thereby ensuring the health and safety of its animals and the quality of chemosensory research on which Monell relies. By replacing the most obsolete part of the Animal Facility, we will ensure that environmental conditions for the animals will be within the AAALAC accreditation guidelines.