Jay L. Vivian - US grants

gene targeting; transforming growth factors; gene mutation; molecular genetics; phenotype; inhibin; site directed mutagenesis; genotype; point mutation; Xenopus oocyte; biotechnology; laboratory mouse; embryonic stem cell; in situ hybridization;

[unreadable] DESCRIPTION (provided by applicant): A variety of mutagenesis techniques in the mouse are available to understand gene function and to develop animal models of human disease. However, the mouse genetics community has lacked a broadly applicable means of performing phenotypic screens to identify mutations in specific biochemical or genetic pathways. The development of cell-based phenotypic screens in mouse embryonic stem (ES) cells could provide the critical methodology for these types of focused mutation screens. The TGF-beta-related signaling pathways would be excellent candidates for directed phenotypic screens in ES cells. These signaling pathways play a critical role in a variety of disease states, including tumor progression. The diverse roles that these signaling systems play in both embryonic development and disease suggests a complex mode of regulation. The nature of how these signals are regulated in an in vivo context is still largely unknown. A directed mutation screen of TGF-beta-related signaling processes would generate valuable animal models to identify and further understand the regulators of these pathways. Many of the components that mediate TGF-beta signaling are present on mouse chromosome 18, suggesting the possibility of a chromosomal-directed phenotypic screen for TGF-beta signaling mutations. In this proposal, reagents will be developed to test the hypothesis that a mutation in mouse ES cells can be identified based on the altered activity of the mutant gene product, and that this altered activity can be assayed using a downstream reporter. A screening strategy will be developed for identifying ES cells that carry chemically induced mutations in the immediate early response components of TGF-beta superfamily signaling on mouse chromosome 18. A luciferase based reporter will be used as a readout of the responsiveness of ES cells to TGF-beta signaling. Mutagenesis will be accomplished with ethylnitrosourea, a highly effective mutagen in ES cells. To resolve recessive mutations, a Cre-loxP-mediated mitotic recombination system will be utilized to generate cells that are homozygous for a mutagenized chromosome 18. Cell-based mutation screening strategies as developed in this proposal can be applied to any active biochemical pathway in mouse ES cells, to generate mouse lines for analysis in vivo. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): High-throughput genome screening of patients provides hope for identifying biological mechanisms in rare, undiagnosed disorders, and may provide broader insight into more common disease categories. However, variant identification is only a first step; a cause-effect relationship between variant and phenotype must be established, and therapeutic strategy formulation requires a thorough understanding of cellular and protein pathologies. The NIH Undiagnosed Disease Program recently characterized a male patient, UDP1757 with neonatal onset of motor dysfunction, cognitive deficiencies, low brain volume, and peripheral neuropathy, worsening at 15 months, and who died at 10 years of age. Genetic screening showed a hemizygous single point mutation in the BHLHB9 locus (C318R) located on the X chromosome. BHLHB9 has been shown to prevent apoptosis and to promote neuronal differentiation and axonogenesis. We hypothesize that C318R results in a loss- or reduction-of-function mutation that results in increased neuronal cell death and reduced axon outgrowth, hence leading to cortical atrophy and broad-spectrum neural dysfunction. The objective of this proposal is to establish a role of the C318R mutation in abnormal neuronal death and reduced neuronal differentiation and axonogenesis. To do so, we propose in vitro assays involving modifications of the BHLHB9 gene in mouse neural stem cells and patient-derived, neutrally differentiated iPS cells in order to define cellular pathologies. We will additionally use mouse models in which Bhlhb9 mutations are introduced, behavioral phenotype determined, and CNS impact defined anatomically. We anticipate that the mouse will recapitulate the clinical phenotype of UDP1757, and that both cell assays and anatomical imaging will yield data consistent with our hypothesis that BHLHB9 C318R is a loss- or reduction-of-function mutation. A strength is a team of experts with complementary skills in pediatric genetics and rare diseases, genetic engineering, transgenic mouse and iPS cell technologies, rodent behavioral assessment, and neuronal culture and quantitation. Additionally, the proposal draws on the rich core resources of the Kansas Intellectual and Developmental Disabilities Research Center. This Exploratory/Developmental Research Grant Award will make significant contributions by establishing cellular processes responsible for an uncharacterized clinical phenotype, and will provide a basis for subsequent studies using the in vitro and in vivo tools developed in this study to formulate, screen and test therapeutic strategies to ameliorate this and related devastating developmental disorders.

Project Summary / Abstract The use of genetically altered mouse and stem cell models are critical tools for research members of the COBRE Project in Molecular Regulation of Cell Development and Differentiation at the University of Kansas Medical Center. The production and analysis of such models ultimately lead to better understanding of basic mechanisms of differentiation and gene expression, functional genomics of disease progression, and in vivo models for diagnostics and treatment. The Transgenic and Gene Targeting shared resource (TGIF), led by Jay L. Vivian PhD, supports members of COBRE and researchers in the Kansas City area by providing centralized technical services for the production of novel transgenic and gene-targeted rodents and genetically altered pluripotent stem cells. The Facility uses cutting edge methods, state-of-the-art instrumentation, and novel reagents for the generation of these models. The COBRE support of the TGIF allows for the development of specific initiatives in the Facility relevant to these researchers. Recent initiatives include successful integration of emerging technologies, including in vivo genome editing methods (e.g. CRISPR/Cas9 and TALE nucleases) to provide a more rapid means of generating novel mouse models. The integration of these continually evolving methods into the `toolbox' of the TGIF greatly accelerates the development of animal and stem cell models of development and differentiation, while also reducing the overall costs to COBRE researchers.  

Transgenic and Gene-Targeting Shared Resource ? Project Summary Genetically altered mouse models are important tools for the researchers at the University of Kansas Cancer Center (KUCC). The production and analysis of such models ultimately leads to a better understanding of the nature, progression and functional genomics of tumor formation. They also serve as in vivo models for diagnostics and treatment. The Transgenic and Gene-Targeting shared resource (TGTSR), led Jay L. Vivian PhD, supports members of KUCC by providing centralized and comprehensive technical services for the production of novel transgenic and gene-targeted rodents and genetically altered pluripotent stem cells. The TGTSR uses cutting edge methods, state-of-the-art instrumentation, and novel reagents for the generation of these models. The TGTSR, housed at the KUMC campus, has four full time technical staff along with Vivian. The Cancer Center support of the TGTSR allows for the development of specific initiatives in the Facility relevant to cancer research. For example, certain transgenic methods and mutations are particularly relevant to cancer studies, including tissue specific transgene expression, subtle mutations that recapitulate clinically identified variants and somatic mutations and strain-specific nuclear transfer. Emerging technologies, including in vivo genome editing methods (e.g. CRISPR/Cas9 and TALE nucleases) are providing a more rapid means of generating these types of novel mouse models of tumor progression. The integration of these continually evolving methods into the `toolbox' of the TGTSR greatly accelerates the development of animal models of cancer, while also reducing costs to KUCC researchers on all campuses.