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Symposium: Improving Human Nutrition through Genomics, Proteomics and Biotechnologies

Phenotype- and Gene-Driven Approaches to Discovering the Functions of Mammalian Genes¹

Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6420

²To whom correspondence should be addressed. E-mail: johnsondk{at}ornl.gov.

	ABSTRACT

TOP ABSTRACT The phenotype-driven approach A gene-driven approach LITERATURE CITED

 
All of us are involved in discovery science as we pursue the genes, networks, cellular processes and biophysical principles that govern our chosen biological question. For those of us who choose to proceed using plant or animal models to dissect the elements of our favorite biological system, there are many classical and newer approaches available for our use, including two complementary strategies by which the discovery process is proceeding at the Oak Ridge National Laboratory (ORNL). The ORNL has been known for six decades for its investigations of the effects of radiation and chemicals in inducing heritable mutations in mouse germ cells, and for using mouse mutations as tools for the cloning and characterization of mammalian genes. Our history and experience in making mouse models are being applied via these two complementary strategies: 1), a phenotype-driven approach, in which mice carrying random chemically-induced mutations are screened for abnormal phenotypes; and 2) a gene-driven approach in which heritable single nucleotide polymorphisms (SNP) in preselected genes already thought likely to influence a biological system of choice can be recovered in live mice. The SNP-carrying mice can then be phenotyped for alterations in one’s target biology. Both approaches have value and are necessary; while we can use mutations in genes that we already know to be of interest in our favorite biology to discover gene function, we also know that biology is full of surprise genes whose effects on our favorite biology would not be predicted and which will be identified only through phenotype screening.

KEY WORDS: • N-ethyl-N-nitrosourea mutagenesis • genotype • phenotype • Tennessee Mouse Genome Consortium • Cryopreserved Mutant Mouse Bank

	The phenotype-driven approach

TOP ABSTRACT The phenotype-driven approach A gene-driven approach LITERATURE CITED

 
The analysis of phenotypes in mutant mice has been the foundation of our understanding of gene function in mammals for more than a century, and has led to the cloning of many genes that when mutated, for the most part, gave rise to easily discernible phenotypes. If we could assume that ablation or alteration of any gene in the genome would have some biological consequence for a mammal, it would be a task of impossible complexity to inspect, test or challenge the animal for any possible structural, behavioral, biochemical or adaptability impact. However, given current and expected insights into categories of gene function and a comprehensive set of high level, high throughput screening tools, it is possible to make quite respectable inroads into recognizing broad classes of abnormal phenotypes amenable to in-depth biological/genetic analysis. Certainly no single program can hope to develop a comprehensive phenotyping pipeline, but with input from each biological interest group in developing broad-level screens and assays, mutagenesis and phenotyping programs will make a good start.

The Oak Ridge National Laboratory (ORNL)² is a founding member of the university-based Tennessee Mouse Genome Consortium, a phenotyping pipeline funded by an NIH coalition led by the National Institute of Mental Health (NIMH) and designed to discover abnormalities of behavior and the central nervous system. Using a three-generation breeding scheme for the recovery of recessive mutations modeled after the Drosophila "balancer" chromosome approach (1), ORNL is inducing single nucleotide polymorphisms (SNP) with N-ethyl-N-nitrosourea (ENU) and producing test-class mice homozygous for the mutagenized chromosome. The test class, the genotypic class to be phenotyped, is recognized by a visual inspection (coat color or ear morphology, for example), and each mutant animal can be expanded into a population of any desired size of like animals for multianimal, multisite and multiscreen phenotyping. In the NIMH-funded effort, test-class mice from all pedigrees undergo a large set of behavioral and neurohistological screens, and then are aged to 18 mo for re-screening for late-onset phenotypes. Ascertainment of an abnormal phenotype in all test-class mice strongly suggests that the mutation maps within the region defined by the chosen balancer chromosome, and heritability is confirmed by breeding tests. Mapping of the phenotype using standard linkage analysis or, for more challenging statistically defined variations, by deletion-complementation mapping into subintervals of manageable size (2), lead to the cloning of the genetic change responsible for the abnormal phenotype.

Is the phenotype-driven approach productive? The ASNS will be interested in a brief description of a mouse model for increased body fat, insulin resistance and glucose intolerance, hyperlipidemia and fatty liver disease. Dr. Madhu Dhar of the University of Tennessee, pursuant to a observation of increased body size in a strain of ORNL mice carrying a chromosome 7 mutation, has discovered that the P-type class V ATPase, Atp10c, is the candidate gene for this interesting set of lipid metabolism abnormalities (3).

	A gene-driven approach

TOP ABSTRACT The phenotype-driven approach A gene-driven approach LITERATURE CITED

 
With the sequence of the mouse genome now in hand, we can design primers for PCR to amplify any regulatory or coding sequence of choice. This capability provides two kinds of opportunities. First, given a list of genes that reside within a delimited interval into which an ENU-induced phenotype has been mapped, each gene can be scanned quickly for an induced SNP responsible for the abnormal phenotype. Second, given a list of genes likely involved in one’s biological system of interest, in invertebrates or vertebrates, we can access by PCR ORNL’s Cryopreserved Mutant Mouse Bank (CMMB), which contains RNA and DNA from 4000 F₁ male C57BL/6JRn mice that carry a load of paternally induced (ENU) mutations. ENU causes SNP in DNA, some of which will occur in functionally important locations in genes. By screening all 4000 samples of the CMMB, one is likely to find an average of about 10 samples that carry a SNP in the DNA sequence of interest. We have already developed a high throughput screening capability for identifying the subset of SNP-bearing DNA sequences among the vast majority of wild-type DNA sequences. Associated with each of these 4000 DNA samples is a cryogenically preserved sperm sample, allowing the recovery by in vitro fertilization or intracytoplasmic sperm injection of progeny carrying the desired mutation identified in the F₁. Thus, the CMMB is a source of induced heritable SNP in the regulatory regions and coding sequences of any gene of choice, throughout the genome, that can be recovered in a live mouse. Our ultimate goal is to operate in a time span in which one can go, in ten weeks, from selecting a gene of interest to having one or more lines of mice heterozygous for ENU-induced mutations in that gene.

The CMMB is in current use, and a number of mutant alleles have been isolated for genes chosen for a variety of interests. We use a high throughput system based on heteroduplex analysis in a 96-well format to identify potential SNP in the chosen DNA sequence, followed by confirmation by DNA sequencing (4). Depending on the location of the mutation within the target gene and on the specific base-pair substitution, ENU-induced mutations may result in frameshifts, single amino acid (missense) changes, exon skipping, etc., thereby producing an allelic series of mutations in any gene. In most cases, multiple alleles are identified, offering the possibility of an allelic series in live mice for the gene of choice so that phenotypic consequences of a series ranging from nulls to slight hypomorphs may often be experimentally determined. Assuming that most human differences likely result from minor rather than major gene expression alterations, such allelic series for one’s genes of choice will be a major resource for laboratories interested in how genetic and environmental factors interact to a whole-organism phenotype. We hypothesize that hypomorphs or expression-level variants for these genes will be more useful than often-lethal knockouts and better models for human genetic variation, and will also provide a protein product for structure/function and biophysical analysis.

Is the gene-driven approach productive? Drs. Ed Michaud and Cymbeline Culiat of ORNL have completed a scan of 7 Mb of CMMB genomic DNA in which a set of ENU-induced mutant phenotypes have been paired with the candidate genes that reside in the region to find which gene has caused which abnormality. In addition, the CMMB is being accessed for mutations in genes of interest to ORNL staff as we build and develop the throughput capability necessary for this resource to become a real "factory" available for the wider community.

ORNL is poised, with phenotype-driven and gene-driven approaches in production mode, to make a significant contribution to the pool of mouse strains available for study by specialists in any field of biology.

	FOOTNOTES

 
¹ Presented at the Experimental Biology Meeting, April 11–15 2003, San Diego, CA. The symposium was sponsored by The American Society for Nutritional Sciences and supported in part by an educational grant from Nestlé and a USDA-NRI conference grant. The proceedings are published as a supplement to The Journal of Nutrition. This supplement is the responsibility of the guest editors to whom the Editor of The Journal of Nutrition has delegated supervision of both technical conformity to the published regulations of The Journal of Nutrition and general oversight of the scientific merit of each article. The opinions expressed in this publication are those of the authors and are not attributable to the sponsors or the publisher, editor or editorial board of The Journal of Nutrition. Guest Editors for the symposium publication are Naima Moustaid-Moussa and Jay Whelan, Department of Nutrition, The University of Tennessee, Knoxville, TN.

³ Abbreviations used: CMMB, Cryopreserved Mutant Mouse Bank; ENU, N-ethyl-N-nitrosourea; NIMH, National Institute of Mental Health; ORNL, Oak Ridge National Laboratory; SNP, single nucleotide polymorphism.

	LITERATURE CITED

TOP ABSTRACT The phenotype-driven approach A gene-driven approach LITERATURE CITED

 

1. Rinchik, E. M. (2000) Developing genetic reagents to facilitate recovery, analysis, and maintenance of mouse mutations. Mamm. Genome 11:489-499.[Medline]

2. Rinchik, E. M., Carpenter, D. A. & Johnson, D. K. (2002) Functional annotation of mammalian genomic DNA sequence by chemical mutagenesis: a fine-structure genetic map of a 1- to 2-cM segment of mouse chromosome 7 corresponding to human chromosome 11p14–15. Proc. Natl. Acad. Sci. USA 99:844-849.[Abstract/Free Full Text]

3. Dhar, M. S., Webb, L. S., Smith, L., Hauser, L., Johnson, D. & West, D. B. (2000) A novel ATPase on mouse chromosome 7 is a candidate gene for increased body fat. Physiol. Genomics 4:93-100.[Abstract/Free Full Text]

4. Li, Q., Liu, Z., Monroe, H. & Culiat, C. T. (2002) Integrated platform for detection of DNA sequence variants using capillary array electrophoresis. Electrophoresis 23:1499-1511.[Medline]

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