Life Sciences Division
1997-98
Progress Report
- Overview
- Mission
- Structure
- Lab Animal Resources
- Operations & Support
- Mouse Genetics Research
- DNA Sequencing Lab
- Gene Expression Chip Lab
- Dosimetry & Biokinetic
- Life Cycle Analysis
- Environmental Assessment
- Biological Sciences
- Functional Genomics & Proteomics
- Structural Biology
- Biomedical Engineering
- Technology Applications
Mammalian Genetics and Development Section
Staff
|
N. L. Cacheiro |
R. S. Foote1 |
W. H. Lee1 |
C. D. Stringer1 |
Technical Support
|
P. R. Akers1 |
K. L. Drake1 |
B. G. Stanford |
Administrative Support
|
J. A. Cripps |
B. P. Warren |
Postdoctoral Fellows
|
H. Chao |
M. Dhar |
B. Jones |
R. Miltenberger |
Subcontractors/Consultants
|
L. Easter |
Students/Faculty/Visiting Scientists
|
M. A. Handel |
A. Schultze |
J. Wilkinson |
J. Zahorsky |
1Retired or terminated employment with Life Sciences Division
in 1997 or 1998
2On loan to Chemical and Analytical Sciences Division
3Part-time
ORNL established a Mammalian Genetics program soon after World War II specifically to assess the genetic effects of ionizing radiations in mammals. Experiments were carried out to determine both rates of induction of germline mutations and differential sensitivities of germ cell stages to induced genetic and cytotoxic damage in mice. In more recent experiments, a wide variety of chemicals have also been tested for germline mutagenicity. While this program generated the data upon which assessments of human risk following exposure are based, it also produced important findings in basic mammalian genetics and led to the establishment of the largest experimental mouse colony in the world. The current mission of the section is the mutagenesis, genetic mapping, and functional analysis of the mouse as proxy mammal for human genetic studies. New mutations are being made in various regions of the mouse genome using both chemical and molecular mutagenesis strategies. Testing is ongoing for subtle phenotypes that would not traditionally have been recognized, even by very experienced technicians who consistently recognize mutations that alter general health, size and morphology, and normal motor behavior. For example, both existing and newly generated mutants are being evaluated for behavioral and biochemical aberrations. Mice that show abnormalities during any of these tests are analyzed in more detail. Technology is being developed to increase the sophistication, efficiency, and throughput for these behavioral assessments. Various bioactive molecules or byproducts in blood and urine are also being measured to monitor mutations induced in biochemical pathways.
Selected Accomplishments
Program Restructuring. In response to both the announced Health Effects Research funding recompetition and to our own sense of the need for redirection of scientific aims, we have revised our programmatic focus. Historically, we have targeted seven specific mouse genes/genome regions for experimental mutagenesis, a strategy that has allowed the accumulation of massive data sets for understanding the mutagenic potential of agents in the mammalian germline and has provided hundreds of mutant stocks for analysis of gene function. In these mutagenesis experiments, emphasis was on the detection of visible and lethal phenotypes resulting from induced genetic alterations. Additional programmatic funding has been devoted to separate but interactive principal investigator/group leader-driven research projects investigating aspects of the functional biology of specific gene mutations.
Beginning in FY 1999, we will use new mutagenesis strategies to accomplish a genome-wide approach to the induction of mutations as tools for the analysis of gene function. Essential elements will be:
- Chemical mutagenesis within selected genome segments defined by chromosomal deletions and inversions, including development of methods for applying this approach genome-wide.
- Broad-based screening of potential mutants for a wide variety of biochemical, behavioral, and morphological phenotypes.
- Cryopreservation, archiving, and distribution of all mutant stocks, and live maintenance of only that subset of stocks necessary for funded projects.
- Development of in vitro mutagenesis strategies, with emphasis on methods especially useful for generating meaningful changes in complex biochemical pathways.
- Basic research into the biology of gene function; the training of young scientists in the design and execution of biological experimentation will be an important element.
- Close interaction with bioinformatics and structural molecular biology research teams, so that our joint, integrated programs form a comprehensive functional genomics effort.
Role of the Agouti Gene in Cancer.
Scientists in MGDS have been involved in research directed at the agouti gene. The goal of this project was to determine if the mouse agouti gene has a primary role in promoting skin cancer. The mouse agouti gene normally regulates the production of pigments involved in hair coloration. However, dominant mutations in the agouti gene cause the normal agouti protein to be produced at high levels throughout the body, resulting in yellow-haired mice that are obese, diabetic, and have an increased susceptibility to cancer of the skin, liver, lung, mammary gland, and urinary bladder. It is unclear if the agouti protein has a direct role in causing cancer, or if the cancers develop as a secondary consequence of the agouti-induced obesity and diabetes. If the cancers develop secondary to the onset of obesity and diabetes, then the agouti gene is unlikely to provide any new insights into the development of cancer. On the other hand, if the cancers develop as a direct effect of the action of agouti, then the agouti gene can be used as a tool to elucidate aspects of the molecular genetic basis of tumor development. The agouti gene was cloned here at ORNL, making it possible to now resolve this issue.
To determine if agouti has a primary role in the development of skin cancer, transgenic mice expressing high levels of agouti in the skin under the regulatory control of the human keratin-14 promoter (K14-agouti) were used in two-stage skin carcinogenesis experiments. K14-agouti mice are not obese or diabetic, but responded to chemical initiation of the skin with a significant increase in skin tumor prevalence (number of mice with tumors) and multiplicity (number of tumors per mouse) compared to nontransgenic control mice. Additionally, agouti acted synergistically with the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) to increase the cumulative prevalence, multiplicity, and malignant conversion rate of skin tumors, and to decrease the latency period of tumor formation. These data demonstrate conclusively that the agouti gene acts as a tumor promoter in skin carcinogenesis, and does so independent of the confounding factors of obesity and diabetes. These findings raise the possibility that deregulation of human agouti, which is 85% similar to the mouse protein, may also be associated with some types of human cancers.
A Mouse Model for the Human Angelman Syndrome. Children born with Angelman syndrome (AS), also known as the "happy puppet syndrome," suffer from severe mental retardation, motor delay, seizures, and behavioral manifestations such as absence of speech, sleep disorders, unbalanced gait, stiff and puppet-like limb movements, and inappropriate laughter. Mutations in a gene, ubiquitin ligase enzyme 3A (Ube3A), found in human chromosome 15q, have been implicated in causing AS.
Among the radiation-induced mutations generated in MGDS's program is a deletion (p30PUb) in mouse chromosome 7 that includes the mouse version of the AS gene, Ube3A. Since large deletions in human 15q cause 70% of known cases of AS, this similar deletion in a strain of mice allows us to determine which of the physical, mental, and behavioral symptoms characteristic of AS can be studied in mice as well. To date, tests have proven that AS mice are quite below normal in their ability to maintain position on a rotating rod; this test measures balance and coordination. AS mice are also much less active than normal mice in a test for exploratory behavior in an activity-test chamber. Interestingly, the performance of mice on these tests improves somewhat with age, mirroring the gradually improving (up to a point) clinical picture seen in AS children as they mature. Further tests are under way to measure the ability of AS mice to remember and try to avoid an unpleasant stimulus as a model for mental retardation, and to assess their 24-hour biorhythms as a model for sleep disturbance.
We have used dissected mouse brains to show which specific brain regions express the AS gene, an experiment obviously not possible in humans. Ube3a is normally expressed in the cerebellum, the center for motor control, and in the hippocampus, thought to control some kinds of learning and memory. Importantly, in both humans and mice, AS occurs only when the mutant gene is inherited maternally. This genetic phenomenon, known as genomic imprinting, is poorly understood and we hope to gather crucial information not only about the functional causes of AS but also about the mechanisms that control imprinting.
A Novel Gene in The Mouse P Region, Expressed Exclusively in the Central Nervous System, Maps to a Human Chromosomal Region Associated with Mental Retardation. One of the Oak Ridge radiation-induced chromosomal deletions, p8FDFoD, at the pink-eyed dilution (p) locus in mouse chromosome 7, disrupts a gene expressed only in the brain and spinal cord. This gene, named Ihw (included in the human WAGR region) encodes two transcripts that we have cloned and sequenced; the DNA sequences show no similarity to other known genes.
WAGR is a human clinical syndrome, consisting of Wilms' tumor (a lethal kidney cancer), aniridia (absence of the iris of the eye), genito-urinary abnormalities, and mental retardation, caused by the deletion of a group of genes in human chromosome 15. The W, A, and G components map as a group to mouse chromosome 2, but the gene grouping has been separated in the mouse so that the R component is in the p region on mouse chromosome 7. Ihw is one of a small cluster of brain-specific genes that may determine the mental retardation in WAGR patients.
Researchers in MGDS have engineered a transgenic knockout of the Ihw gene, and are breeding mice that will have both copies of Ihw disabled. Those mice will go through our behavior-testing center to determine if the absence of the Ihw gene product causes abnormalites in mice that may mimic the mental retardation associated with WAGR in humans.
Major Cryopreservation Effort for Mutant Mouse Stocks. MGDS has launched a major cryopreservation effort for mutant mouse stocks. By freezing sperm and/or embryos, we propose to become a major archiving and distribution center for experimentally induced mouse mutations that have significant phenotypes of interest to the functional genomics and wider biological communities. Sperm freezing will provide a facile means for distributing any mouse mutation that is not male-lethal or male-sterile to the scientific community, and thawed embryos can very reliably be used to reconstitute many stocks, especially inbred strains, via transfer into recipient females.
Cryopreservation will also provide a logistically feasible means for rederiving the conventional (not pathogen-free) ORNL colony into a new, specific-pathogen-free (SPF) facility, when that facility becomes available within three years. We are endeavoring to freeze down mutant stocks now so that only those stocks that will be actively used upon opening of the new facility need be reconstituted. In the interim before our move into the clean facility, we will cryopreserve many stocks not used in active investigations in order to free our genetics staff for new experimental work.
Since the effort has been running five to seven days a week, we have frozen 3000 embryos; this is more than were frozen in the entire previous year. We have also frozen sperm from 39 mutant stocks, and are collecting mice from an additional 80 stocks into the freezing queue. The effort has included the quality-control measures necessary to ensure that our recovered embryos are fully viable and our thawed sperm competent for fertilization either by artificial insemination or in vitro fertilization. We are also in active collaboration to test the efficacy of freezing whole ovaries for subdivision and transplantation into recipient females; this method would be invaluable for male-sterile and female-subfertile stocks. This collaboration includes testing to determine if embryos, sperm, and ovaries from non-SPF stocks still transmit any potential pathogens when used to reconstitute live stocks.
Identification of Gene Associated with Deafness, Gastritis And Gastric Lymphoma. Researchers in MGDS have determined that the disruption of the mouse mucin 2 (muc2) gene in a unique Oak Ridge mutation called 14Gso produces gastritis and gastric cancer and may also be involved in hereditary deafness.
14Gso is an X-ray induced mutation. Mutant mice were easily identified because they exhibit a persistent circling and head-bobbing behavior, indicative of defects in the inner ear, the organ where balance and hearing are controlled. Direct examination of the inner ear structures of 14Gso mice revealed severe degeneration such as a collapse of membrane structures in the cochlea and vestibular apparatus at birth, progressive deterioration of hair cells, nerve and supporting cells. Culiat and Stubbs conducted research directed toward the identification of the gene(s) responsible for these defects and further biological characterization of the 14Gso mutation.
Cytogenetic analysis showed that 14Gso is a mutation due to the breakage and exchange of the tips of mouse chromosome 7 and 10. Further cytogenetic and physical mapping data of the mutation indicated that the breakpoint region in chromosome 7 is located at the regulatory region of muc2 (intestinal mucin 2), gene coding for a major protein in the mucus lining of the intestine. Since a region of the human muc2 gene was very similar to the gene associated with deafness in man (Norrie Disease Protein), the expression of this gene in 14Gso was investigated. The muc2 gene expression is abnormal in mutant mice, indicating a loss of regulation of the normal levels and tissue specificity. The gene is normally expressed in the intestine and in mutant mice it is found at very high levels in the stomach and also misexpressed in the kidney and lungs. In man, the overexpression of muc2 (could be induced due to infection by the bacterium causing ulcer, Helicobacter pylori) in the stomach is associated with chronic gastritis leading to gastric lymphomas and adenocarcinomas. When stomachs of mutant mice were examined, these same defects were discovered. 14Gso is therefore a good mouse model for studying the progression of gastric cancer from chronic gastritis.
Abnormal expression of muc2 in 14Gso in the inner ear has not been demonstrated; however, it is well known that mucin-like proteins are found in the mammalian inner ear, though their functions are not known. Muc2 is very large and so far only pieces of the mouse gene have been cloned and are useful as probes for expression in the inner ear. The region of mouse muc2 with highest homology to the Norrie Disease protein has been difficult to clone. Cloning and sequencing of the entire gene is currently being done at Lawrence Livermore National Laboratory. The cloning and characterization of the mutated regions in 14Gso (both translocation breakpoints in 10 and 7) will be completed as a collaborative project between LLNL and ORNL.