LELAND STANFORD JUNIOR UNIVERSITY, THE
Grant: $291,941 - National Institutes of Health - Sep. 1, 2009
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Award Description: The long term goal of research in this lab is to understand how sensory experience during critical periods of development, mediated by the activity-driven functioning of circuits in the CNS, is translated into lasting structural change in synaptic connectivity. The specific goal of this project is to examine the hypothesis that activity-dependent synaptic remodeling in development, and synaptic plasticity in the adult hippocampus, involve a family of molecules with well-known function in the immune system: MHC Class I genes (the HLA genes in humans). Research in this lab has revealed a new and unexpected role for these 'immune molecules in the healthy CNS. Two experiments are proposed, both with the goal of elucidating specific neuronal roles for the classical MHCI genes H2-Kb and H2-Db in the regulation of synaptic plasticity and morphology. To accomplish this goal, loss of function (knock out) , rescue of function, and overexpression experiments in the same tissue and cell types are needed. Experiment #1 will generate a transgenic mouse carrying a conditional allele of H2-Db. Experiment #2 will perform rescue and overexpression experiments using GFP-tagged H2-Db and H2-Kb expression constructs and by generating Lentiviral vectors for use both in hippocampal neurons in vitro and in CNS structures in vivo. It should be possible to generate the vectors and the transgenic mouse within the requested one-year funding period since experiments are already under way. The results of these experiments should broaden understanding of how use-dependent changes, both in development and in adulthood, are encoded in the structure of neural circuits. Moreover, because a neuronal MHCI is known to be modulated by inflammation and can be recognized by cells of the immune system, this research has the potential to provide a direct explanation for how the nervous and the immune systems communicate with each other in both health and disease. Changes in synapses and circuits occur during critical periods of learning in childhood, as well as in memory formation throughout life. Understanding molecules and mechanisms involved in this process is also crucial for addressing and ultimately curing disorders of learning and memory, from Dyslexia and Autism in childhood to Alzheimer’s and other memory disorders of the aging brain.
Project Description: Status report on experiment #1, cloning of a targeted conditional mutant allele of the H2-Db gene: Progress has been made on engineering a targeting construct for the conditional H2-Db knockout mouse. A 13kb genomic fragment that contains the H2-Db gene locus and surrounding sequences will be subcloned. Recently, we prepared a receiving plasmid with homologous sequences to the ends of this 13kb genomic region that will be used for subcloning by homologous recombination in E.coli. Additionally, we have generated and sequenced PCR products corresponding to regions in introns 1, 2, and 3, and exons 2 and 3, and have inserted a LoxP site into intron 1. Currently in progress is insertion of a Frt-Neo-Frt-LoxP cassette into intron 3. This small fragment of genomic H2-Db DNA will then be digested at endogenous restriction sites, and cloned into the corresponding positions in the 13kb genomic DNA fragment. The resulting plasmid will contain the LoxP sites flanking exons 2 and 3 and a Neo cassette for selection in ES cells and will be used as the targeting construct.
Jobs Summary: No jobs created or retained yet. (Total jobs reported: 0)
Project Status: Less Than 50% Completed
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