Andras Nagy - US grants

DESCRIPTION (provided by applicant): This project will develop new tools to facilitate high throughput, genome-wide mutagenesis in mouse embryonic Stem (ES) cells. Functional analysis of the mouse genome is a critical component of functional genomics, providing information relevant to human gene function and disease states. Gene trap mutagenesis in ES cells is a proven technology for generating sequence-tagged insertional mutations in mice. We propose to develop new gene trap vectors, incorporating a novel integrase-mediated recombination system. This vector will be used to generate a library of ES clones containing insertions within a wide range of genes across the genome. This library will be screened for expression via in vitro expression assays, using differentiation protocols already developed in the group. Each insertion will also have a RACE sequence tag associated with it and all information will be incorporated into an online database for availability to the community. Interesting insertions can be used to study mutant phenotype after germ line transmission, or can be used as sites of further gene modification or addition, by means of the integrase system. A complementary strategy of mutagenesis in ES cells will also be developed, focusing on in vitro recessive phenotypic screening, combining chromosome-specific loss of heterozygosity with chemical mutagenesis of ES cells. Both strategies will be developed first in existing 129 ES cells, but will be transferred to newly derived C57BL/6 cell lines, as soon as they are validated, to provide mutations on the B6 'gold standard' background.

[unreadable] DESCRIPTION (provided by applicant): The overall objective of this project is to further the value of the mouse as a powerful and important tool in the study of human disease through the establishment of a publicly available, comprehensive collection of a fully C57BL/6-based null resource knockout embryonic stem cell lines, from which a library of mice containing a null mutation in every gene in the mouse genome can be developed. This application proposes to establish an international consortium of academic institutions comprised of four Canadian (Drs. Nagy, Hicks, Ding and Rancourt), one German (Dr. Wurst) laboratories and the University of Missouri Comparative Medicine Center (Dr. Critser). This consortium was developed to address the NIH RFA (RFA-HG-05-007) for the production of a comprehensive resource of mouse mutants in which every gene in the mouse genome has been knocked out by a null mutation marked with a reporter system of high utility. This consortium fulfills the criteria for an ideal resource in that all of the mutations will be carried on a uniform background strain, C57BL/6, which is the strain most widely utilized by mouse researchers. The unique aspect of this overall proposal is that one laboratory in the consortium (Dr. Nagy's laboratory) has established an exceptional C57BL/6 embryonic stem cell line with a very high germline transmission (~70%). Importantly, this consortium has members which are also part of a Canadian funded project (NORCOMM; Nagy, Hicks, Ding and Rancourt labs) and a European Union funded project (EUCOMM; Wurst lab) to perform high throughput gene mutations in embryonic stems cells. Combining the existing experience and expertise of three major laboratories making null mutations, in combination with the Nagy lab's high germline efficient C57BL/6 embryonic stem cell line and the University of Missouri's expertise in creation of mice and operation of quality control/assurance programs for large NIH-funded resources; will provide a highly efficient and cost effective "team" to achieve the goals of this RFA. While several consortium members are involved with similar world-wide efforts to provide a complete library of mouse null imitations, there is no overlap between those projects and this KOMP initiative. In fact, a major strength of this proposal is the vast experience and existing infrastructure in those laboratories. The existing high throughput, low cost methods that are "up and running" in our collective laboratories enable this consortium to accomplish the goals of the KOMP in an extremely timely and cost efficient manner. Importantly, because this consortium is comprised of academic groups, in which there are multiple, separately funded, ongoing research projects directly related to the KOMP, we will be able to ensure continuous access to, and application of, the most cutting-edge technologies and science; thereby providing continuous quality improvement over the course of this KOMP. This aspect will ensure the most cost effective methods are applied at each step along the way to completing the core goals of the KOMP. [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The utility of transgenic tools that can selectively label each particular type of neuron, and furthermore, selectively inactivate genes or manipulate the activity of one type of neuron at a time, has high impact in many different areas of neuroscience research. Current approaches taken by many, e.g. the Cre-driver line creation, usually utilize genes relatively specifically expressed in certain cell types. However, such "marker" genes rarely label a really specific neuronal population. It is highly desirable to further refine the target specificity into a particular region or a specific population of neurons. To achieve this we propose to systematically evaluate a number of combinatorial strategies and develop a series of transgenic tools to drive highly specific gene expression through the intersection of the expression of 2 marker genes. Through mining our genome-wide database, Allen Brain Atlas, Dr. Zeng and colleagues have identified a large set of gene markers for different cortical cell types. Dr. Nagy has developed a set of gene targeting and allele replacement strategies for expressing multiple genes efficiently. Allen Institute has a high throughput ISH platform and capability of high resolution image acquisition and databasing. By combining the superb technologies developed by both parties, we will examine the colocalization of different marker genes by double fluorescent ISH (dFISH), create both driver and reporter/responder mouse lines to test different intersection strategies, and use the dFISH again to systematically characterize and database where the controlled gene expression occurs in the entire mouse brain. In this grant we will use the neocortex as a model to establish the technology. Once completed, it can be applied easily into other brain regions as well. PUBLIC HEALTH RELEVANCE: Studying the extreme complexity of the brain demands ways to tease apart its components. Traditional transgenic tools have been widely used in every area of neuroscience research. Our proposed research will provide two types of powerful tools to the neuroscience community: transgenic mouse lines that can direct genetic manipulation to highly specific neuronal populations, and an image database comprehensively documenting where such manipulation occurs. Both of these tools will have major impact on the broad neuroscience community, from molecular to systems neuroscience, from development to behavior studies, from studies of normal brain physiology to disease mechanisms. [unreadable] [unreadable] [unreadable]