James M. Pickel, Ph.D. - US grants

Creation of disease models: [unreadable] [unreadable] Human disease models for two neurological disorders were developed by the core facility over the last year. Because the critical genes involved in these diseases has been defined the mouse gene could be identified and modified to match the genetic change that leads to human suffering. These models pave the way for the etiology of the disease and the development of therapies to ameliorate the human disorder. In addition the study of these disease models can elucidate--through their disruption--the basic mechanisms of neural function and development.[unreadable] [unreadable] Mucolipidosis IV is a disorder caused by any of several genetic alterations in the Mcoln1 gene. Patients have developmental delay, corneal clouding, retinal degeneration, hypergastrinemia, achlorohydria as well as some poorly defined brain anomalies. On the cellular level inclusion bodies are found in many tissues. The MCOLN1 protein appears to form a channel that is critical for the function of lysosomes. The mouse model of this disease was created by targeting the Mcoln1 gene in ES cells that were selected and screened for the expected disruption of the gene. Mice were produced from the ES cells and were screened for the absence of mRNA transcripts. Three cell lines produced mouse lines with identical phenotypes. Initially the null animals appear normal, but as they mature there are evident changes in body composition. The animals appear thinner and eventually develop postural changes. Hindlimb strength is progressively lost until paralysis is complete. Eye phenotypes, increased blood gastrin and inclusion bodies are also present. Seven different laboratories have been studying these animals, which will soon be made available generally as the work is published.[unreadable] [unreadable] Familial dysautonomia effects sensory and autonomic nerves and causes a range in severity of symptoms from temperature regulation to control of stomach reflux. The mouse model is made more complex since the null embryos die before birth. The strategy has been to replace the mouse gene with human transgenes of either the normal or disease-causing allele. The number of copies of these transgenes that are incorporated into different mouse lines has been critical to mimic the disease. Now that those technical issues have been addressed it is possible to treat mice with a compound that has been demonstrated to reverse some biochemical effects of FD in patients' cultured cells. This raises the possibility that a treatment for patients can be discovered.[unreadable] [unreadable] These models of genetic disease were developed in the core facility and have been analyzed by laboratories that specialize in each of the aspects of the disease phenotype that is mimicked in the mouse model. Now as this work is published these mice will be distributed freely to laboratories for broader evaluation. [unreadable] [unreadable] Production of transgenic mice[unreadable] [unreadable] The NIMH transgenic core uses most of its resources to create transgenic animals and provide other transgenic animal services to neuroscientists at NIH. This allows NIH scientists to test models of disease, investigate the role of specific genes in behavior and the development of the nervous system. Scientists at NIH can quickly apply basic research directly to an intact organism. Over the last year (FY2007; September 2006-October 2007) the core facility has worked on more than 65 different transgenic projects. Of these 31 used randomly integration transgene and were generated by injecting the purified gene into a fertilized mouse oocyte. Thirty three of the lines carried targeted transgenes that were first integrated into ES cells (mouse embryonic stem cells) which were selected and screened for the proper recombination event before being expanded and injected into mouse blastocysts. Both techniques generate mice with an altered genome: with genes either added, deleted or altered. In addition to the transgenic mice that were produced the core facility archived 20 different mouse lines by cryopreservation. Fifteen different transgenic lines were rederived: that is mice from these lines were used to produce embryos that could be transferred into the specific pathogen-free animal facility in building 49 of the NIH campus in Bethesda.[unreadable] All of these projects are undertaken as a service and will be described in the reports from individual investigators. But they can be categorized into general areas: [unreadable] 1) many projects exploit the use of recombinases to effect genetic changes in specific temporal and spatial compartments. Several investigators have, and continue to request animals that express the cre recombinase in specific patterns that can then be used to alter expression of genes in defined loci at specific times. Some of these projects require the screening of multiple lines to find those that express in the desired pattern. [unreadable] 2) The GENSAT project uses this same approach. The core has archived and distributed several GENSAT lines that have been evaluated and found to be useful for neuroscience research.[unreadable] 3) Other projects have used inducible transgenes to allow expression at specific times and in limited areas. _[unreadable] 4) Beside these efforts to extend the utility of transgenic techniques there are many projects in which genes are disrupted or expressed in aberrant patterns to gauge the effect on behavior. Genes that have been implicated in schizophrenia and bipolar disorder and other innate behaviors have been manipulated for NIMH investigators.[unreadable] 5) Many of the other projects that have altered expression of molecules that have poorly defined function but are expressed in the nervous system in interesting patterns that suggest a role in learning, memory or development.[unreadable] 6) And finally several projects have used inducible or specifically expressed toxins to ablate neural cells to see what roles these specific cells play in the function of the brain.[unreadable] [unreadable] Technology development[unreadable] [unreadable] Derivation of embryonic stem cell lines [unreadable] The genetic background of research mice has a significant effect on behavior, as well as the basic anatomy, development and function of the nervous system. Inbred strains of mice have been created to provide a homogeneous genetic background upon which changes due to experimental perturbations can be observed. The technology of producing transgenic mice relied, historically, on outbred or poorly inbred strains. Recently the use of inbred strains in transgenic production has increased. In the NIMH transgenic core new ES lines have been developed from mixed and inbred strains. In addition all transgenics are now produced on a C57Bl/6 background to decrease the time required to backcross a transgene into this universally utilized strain. In addition these ES lines ubiquitously express the green fluorescent protein (GFP) that can be used to track viable cells from the embryo through maturity. The ES cells that were derived from these efforts were distributed to other groups and most recently were licensed for sale.[unreadable] Other projects to increase efficiency of transgenic production.[unreadable] Several efforts to increase the efficiency of transgenic mouse production have been initiated in the core facility. The ES cell lines mentioned above have been a major improvement over other lines that are available. Also, methods of purifying transgence DNA for injection or electroporation have been optimized over the last year. In vitro fertilization (IVF) methods have been used to rescue valuable transgenic lines that are no longer fecund. Production of mice from ES cell / blastomere aggregates have been used. When coupled with tetraploid blatomeres this results in a mouse that is derived solely from ES cells. And most recently alternatives to blasocyst injection have been developed. By injecting ES cells into the morula stage embryo we hope to improve the incorporation of ES cells into the resulting mice.

The potential associations between human genes and mental disorders are being increasingly identified. But with these associations comes a conspicuous lack of understanding of how these genes function in an afflicted, or even a presumably normal brain. By creating animals with the same genetic changes and even some carrying human disease genes these associations can be tested. By producing mice with human disease alleles neuroscientists can study the behavioral, developmental, anatomical, cellular and biochemical levels of the disease. From this approach the normal function of these genes can be defined. This approach has been used to provide animal models for many research projects at the NIH IRP as well as with collaborators in the extramural program. The details of these experiments are described in investigators own reports. Below is a partial list of our research projects that suggests the scope of the areas of investigations that have benefited from animal models produced by the core facility. These projects cover a wide range of neuroscience experiments at the level of specific molecules, gene expression, cell biology, neural circuits, learning, complex behavior, and include studies of specific diseases. Stress: the role of a specific gene (catachol-O-methyltransferase) in the susceptibility to stress was demonstrated in mice that were engineered to have reduced levels of this gene. Learning and memory: In the past year transgenic mouse models have been used to show the role of specific protein synthesis on learning and memory. Other transgenic mouse models have been used to show the role of specific peptide-expressing cells to influence the link between fear and behavior and learning. Neurogenesis: From mid-gestation and into old age, new neurons are produced in the brain. The role of these new cells that appear in adults is especially interesting, and suggests a function in learning and memory and potential treatments for neurodegenerative disorders. Mucolipidosis IV: The mouse model of this disease resulted from a long-standing collaboration with the Slaugenhaupt laboratory and has continued to yield results, including a description of the neuropathy that may be associated with this disease. The core facility continues to distribute these animals. Familial dysautonomia: Another collaboration with the Slaugenhaupt lab resulted in a model for this disease. The lines that carry either a human normal or disease gene are being created in the core. Those are crossed into a null line to replace the endogenous IKBKAP gene with its human disease equivalent. The core produced several new lines this year to test the effect of multiple copies of the gene. Activity in glial cells: The core produced a mouse line with a transgene that indicated the concentration of calcium in glial cells. By changes in its fluorescent properties the calcium concentration, and associated activity, has been demonstrated in these cells. Manipulating circuitry: Mice in which specific neurons could be rendered transiently inactive have been produced for two separate laboratories. Those laboratories are investigating different neural circuits that are active in learning and addiction. Reporter and effector mice: Several lines that express effector molecules like CRE recombinase at specific temporal and spatial compartments were produced. Other lines that were used to report the activity of these and other recombinases were created. Mice, such as those from the Gensat project were rederived and exported. Transgenic rats: Technological development projects have allowed the core to produce transgenic rats for investigators in three NIH intramural institutes.

SUMMARY The NIMH transgenic core facility has several major functions: 1) to produce transgenics for neuroscience research at NIH 2) develop new transgenic techniques and model systems 3) support research with associated techniques in genetic research in neuroscience and 4) engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Meterics of production over the past year included: a) 48 transgenic mouse lines produced by oocyte injection. b) 14 rat lines have been produced by oocyte injection. c) 13 mouse projects have first altering the genes of embryonic stem cell and then using those to produce mice. 2) Development Over the last year projects to allow more productive methods have been developed. Two notable projects are a) development of a panel of rats that produce CRE recombinase under the control of transcriptional promoters that are specific for subpopulations of neurons. b) developing methods to produce transgenic marmosets. Over the past year rapid progress in developing techniques to produce transgenic marmosets have been made in the core facility. The methods to produce an excess of embryos, to optimize surgical procedures to harvest those embryos most efficiently, to culture those embryos, develop transgenic lentivirus, infect embryos, transfer those embryos into recipient females using ultrasound visualization and develop tissue culture methods for primary marmoset cells have been developed. This extensive effort has set the stage for several projects for NIH researchers to use these transgenic animals in basic research in the intramural program. 3) Technical Support a) 98 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 68 lines have been rederived, by transferring lines from pathogen bearing animals into those with defined health status. c) transgenic project design and assistance have continued to be significant to NIH neuroscience labs without experience in producing transgenic animals. 4) Collaborative projects: below is a list of projects that have been initiated in 2011, or have continued from last year. Stress: The role of a specific gene (catachol-O-methyltransferase) in the susceptibility to stress was demonstrated in mice that were engineered to have reduced levels of this gene. Neurogenesis: Transgenic mice and, more recently, transgenic rats have been generated to study the role of neurogenesis in adults. From mid-gestation and continuing into old age, new neurons are produced in the brain. The role of new cells appearing in adults is especially interesting, and suggests a function in learning and memory and potential treatments for neurodegenerative disease. Schizophrenia: Mice with behavioral characteristics that resemble schizophrenia were produced by mating effecter mice from the core facility with responder mice. The latter carried a conditional ablation of the NMDA receptor. The offspring of these matings were engineered to lack NMDA receptors in a subset of corticolimbic interneurons. They displayed behavioral deficits in normal mating, nest-building and anxiety-like behavior. Memory dysfunction was also revealed in these animals. Stress and neurogenesis: A recent paper using mice developed in the transgenic core has shown that neurogenesis is critical for maintaining a normal response to stress. This finding is significant because it conlusively identifies this role for neurogenesis in adult animals. Following other work that shows that stress reduces neurogenesis, this new finding implies a cycle in which stress can increase. Learning and memory: The effect of specific and tightly controlled protein synthesis on learning and memory was studied. In addition, transgenic mouse models have been used to show the role of specific peptide-expressing cells to influence the link between fear and behavior and learning. Manipulating circuitry: Mice have been produced for two separate laboratories which have specific neurons that could be rendered transiently inactive by light activated ion channels. Those laboratories are investigating different neural circuits that are active in learning and addiction. Drug addiction: Lines of transgenic rats that express GFP in response to afferent input activation of the fos gene were generated in the core facility. These rats are being used by Bruce Hopes laboratory in NIDA to study patterns of neural activity in response to addictive drugs. . Mucolipidosis IV: The mouse model of this disease resulted from a long-standing collaboration with the Slaugenhaupt laboratory and has continued to yield results, including a description of the neuropathy that may be associated with this disease. The core facility continues to distribute these animals. Familial dysautonomia: Another collaboration with the Slaugenhaupt lab resulted in a model for this disease. Lines carrying either a human normal or disease gene are being created in the core. These lines are crossed into a null line to replace the endogenous IKBKAP gene with its human disease equivalent. Glial activity reporters: The core produced a mouse line with a transgene that indicated the concentration of calcium in glial cells. By changes in its fluorescent properties, the calcium concentration and associated activity has been demonstrated in these cell. Reporter and effector mice and rats: