Functional Genomic Studies of Early Myogenic Differentiation

早期肌源分化的功能基因组研究

• 批准号: 7663827
• 负责人: Joseph C Glorioso
• 金额: $ 31.67万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7663827
• 关键词: A Mouse; Adult; Aliquot; Appearance; Bacteria; Bacterial Artificial Chromosomes; Biochemical; Biological Assay; Biological Models; Candidate Disease Gene; Cell Differentiation process; Cell Line; Cell Lineage; Cell division; Cells; Cloning; Cloning Vectors; Code; Commit; Complement; Complementary DNA; Complex; Condition; DNA Binding; DNA Library; Data; Databases; Defective Viruses; Detection; Development; ES Cell Line; Early Promoters; Eating; Elements; Embryo; Engineering; Enhancers; Essential Genes; Event; Excision; Expression Library; Figs - dietary; Fluorescence; Gene Expression; Gene Targeting; Genes; Genetic Recombination; Genome; Goals; Growth; Growth Factor; HSV vector; Herpesvirus 1; ICP47; IGF1 gene; Image; Immediate-Early Genes; In Vitro; Infection; Insulin-Like Growth Factor I; Joints; Kinetics; LIF gene; Left; Libraries; Life; Methods; Modeling; Mus; Muscle; Muscle Cells; Muscle Development; Muscle satellite cell; Nature; Pathway interactions; Phenotype; Plasmid Cloning Vector; Plasmids; Play; Polymerase Chain Reaction; Population; Process; Production; Proteins; RNA; Range; Reporter Genes; Research Personnel; Role; Serum; Signal Transduction; Simplexvirus; Staging; Standards of Weights and Measures; Stem cells; System; TK Gene; Technology; Testing; Time; Tissues; Trans-Activators; Transcriptional Regulation; Transplantation; VP 16; Vero Cells; Vertebral column; Viral; Viral Proteins; Virion; Virus; Virus Replication; base; cDNA Expression; cDNA Library; cell growth; cellular engineering; cytotoxic; day; embryonic stem cell; engineering design; experience; functional genomics; insight; interest; leukemia inhibitory factor; mutant; myogenesis; nectin; novel; particle; progenitor; promoter; protein expression; receptor; red fluorescent protein; research study; stem; transgene expression; vector

The overall goal of this project is to employ a novel functional genomics approach to identify genes whose products induce the differentiation of mouse embryonic stem (ES) cells towards muscle cells. In Aim 1, a highly defective, non-cytotoxic HSV mutant will be developed that is deficient for expression of the immediate early (IE) genes and capable of replication in cells engineered to contain a vector-inducible, trans-complementing ICP4 gene. In Aim 2, a panel of mouse ES cell lines will be created in which either the EGFP or ICP4 gene will be recombined into the cellular genome juxtaposed to the promoter that drives expression of one of the muscle differentiation factors Pax7, myf5, or myoD. Using established culture conditions, the EGFP cell lines will be differentiated towards muscle cells and the appearance of fluorescing cells will be correlated with detection of stage specific myocytic markers. In Aim 3, the mutant virus will be engineered to contain a bacterial artificial chromosome and a recombination system, "Gateway" (Invitrogen), which will then be used to introduce a mouse muscle lprogenitor cell cDNA library into the vector. The vector library will be propagated in bacteria as single lcopy plasmids, its complexity established by quantitative PCR for low and medium copy genes and ltransfected into ICP4-complementing Vero cells for virus production. In Aim 4, pooled library vectors will lbe propagated on complementing Vero cells in microtiter wells and replica plated onto the three myogenic promoter-ICP4 ES cell lines. Progeny vector particles generated in cells expressing ICP4 by induction of the resident myogenic promoter will be isolated by limiting dilution on complementing Vero cells and rescreened on the panel of EGFP ES cell lines to determine whether the cellular factor is functional in activating early and late stage myogenic factor promoters. The cDNA of activating vectors will be sequenced, compared with known data bases for preliminary assessment of function and further characterized for their ability to induce muscle cell markers using immunological and biochemical assays. Ultimately, we hope to identify novel factors or signaling mechanisms that interact to control early muscle cell development.

该项目的总体目标是采用一种新的功能基因组学方法来识别基因