PROJECT llI; EXPRESSED CDH CANDIDATE GENES CAN BE PREDICTED THEN FUNCTIONALLY

项目III；

基本信息

批准号：
8143192
负责人：
PATRICIA K DONAHOE
金额：
$ 43.44万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8143192
关键词：
Address Affect Algorithms Animal Model Animal Sources Animals Area Bioinformatics Biological Models Birds Candidate Disease Gene Cell Line Collaborations Communities Comorbidity Complement Congenital Heart Defects Copy Number Polymorphism Cytogenetics DNA Resequencing Data Data Set Defect Development Diaphragmatic Hernia Doctor of Medicine Doctor of Philosophy Drosophila genus Dysmorphology Embryo Etiology Gene Dosage Gene Expression Gene Expression Profile Gene Mutation Gene-Modified Generations Genes Genetic Genetic Variation Genome Genomics Heart Homologous Gene Human Immunohistochemistry In Situ Hybridization Injection of therapeutic agent Institutional Review Boards Knock-out Knockout Mice Laboratories Lung Micromanipulation Modeling Modification Molecular Mus Mutation New York Organ Culture Techniques Pathway interactions Patients Phenotype Proteins RNA Interference Research Personnel Resources Respiratory Diaphragm Site Structure Techniques Terminator Codon Testing The Jackson Laboratory Transgenic Organisms Tretinoin Universities Validation Variant Virus Work animal breeding arm base cardiogenesis cohort design exome experience fly gene function high throughput screening in utero induced pluripotent stem cell interest knock-down laser capture microdissection middle column mutant mouse model novel offspring programs promoter protein protein interaction receptor research study small molecule trend vector

项目摘要

In PROJECT III (Expressed CDH Candidate Genes Can Be Predicted, Then Functionally Validated in Animal Models and IPS Cells) we are addressing a most daunting loggerjam facing the genomic and genetics community. In addition to the challenge of priortizating the large data sets emanating from each of the genomic platforms, is to garner the resources and expertise to confirm the functionality and causality of the candidates in which a computationally significant variant is detected. In this project, each variant will be evaluated by stringent criteria that requires that the sequence variants be unreported in dbSNP and the 1000 Genomes Project, and that they are nonsynonymous and cause structural changes in the protein or introduce stop codons (Intertronic variants should affect species). Such variants will then be assessed for testing in the multiple animal model systems uniquely available In the Donahoe laboratory or through our collaborations with The Jackson Laboratory, under the direction of Carol Bult, Ph.D., the Co-PI of Project III, or the laboratory of Co-investigator Kate Ackerman, M.D., at University of Rochester, New York. If a mouse knockout is available and survives beyond El 5.5, we will breed these animals at The Jackson Laboratory and examine the diaphragms for defects equivalent to human diaphragmatic hernia. If the animals do not survive beyond El 5.5 (Right arm of Figure 2), a conditional knockout will be created using a promoter such as NKX3.2 to direct expression to the diaphragm under the direction of Dr. Ackerman. Embryos of these offspring will be examined at E15.5, for defects in the diaphragm. Finding a defect will confirm the functional importance of the expressed gene and make it a candidate in the search for downstream genes which can rescue the phenotype in high throughput screens. If a mouse knockout is not available (Middle column of Figure 2), we are equipped in the laboratory to knock down the expression of a gene of interest by direct in utero injection into RCAS receptor transgenic embryos, the RCAS-TVA vector containing the gene variant. This work will be done in the Expression Core directed by Maria Loscertales, Ph.D., who will also use the avian model to determine in ovo if there is a modification of the lung phenotype. Affected lungs can be then be cultured in organ culture and tested for rescue. Dr. Loscertales will also confirm expression by immunohistochemistry and in situ hybridization, which requires considerable expertise to be done properly in the tiny embryonic diaphragm. She also has considerable experience with micromanipulation techniques to be able to employ this technique data in the mouse embryo at E10 and in the chick in ovo. Dr. Loscertales' expertise is complemented by that of Dr. Kate Ackerman, co-investigator of Project III, who has over six years of experience in identifying the small mouse diaphragm structures and evaluating their dysmorphology.

在项目III中（可以预测表达的CDH候选基因，然后在功能上在动物模型和IPS细胞中得到验证）我们正在处理面对基因组和遗传学界的最艰巨的loggerjam。除了挑战从每个基因组平台发出的大数据集的挑战之外，还要获得资源和专业知识，以确认检测到计算重要变体的候选者的功能和因果关系。在该项目中，每个变体将通过严格的标准进行评估，该标准要求在DBSNP和1000个基因组项目中未报告序列变体，并且它们是非同义词的，并且会导致蛋白质中的结构变化或引入终止密码子（Intertronic变体应影响物种）。然后，将评估此类变体在Donahoe实验室中独特地使用的多个动物模型系统中进行测试，或通过我们与Jackson Laboratory的合作，在Carol Bult，Ph.D.，Project III的Co-Pi，或Rochester，New York Rochester的M.D.的Kate Ackerman，M.D.如果有小鼠的敲除并在EL 5.5以上存活，我们将在杰克逊实验室繁殖这些动物，并检查隔膜是否相当于人diaphragragmatragmatic的疝。如果动物无法生存超过EL 5.5（图2的右臂），则将使用启动子（例如NKX3.2）创建有条件的敲除，以在Ackerman博士的指导下将表达在隔膜上。这些后代的胚胎将在diaphragm中的缺陷中检查在E15.5上。找到缺陷将确认表达基因的功能重要性，并使其成为寻找下游基因的候选者，该基因可以在高吞吐量筛选中拯救表型。如果不可用小鼠敲除（图2的中间列），我们将在实验室配备，通过将子宫注射直接注射到RCAS受体转基因胚胎中，将含有基因变体的RCAS-TVA载体击倒感兴趣的基因。这项工作将在玛丽亚·洛斯塔莱斯（Maria Loscertales）博士指导的表达核心中完成，他还将使用鸟模型在OVO中确定肺表型的修饰。然后可以在器官培养中培养受影响的肺并进行救援。 Loscertales博士还将通过免疫组织化学和原位杂交来确认表达，这需要在微小的胚胎隔膜中正确地进行专业知识。她还具有微观流动技术的丰富经验，能够在E10的小鼠胚胎和OVO中的小鸡中使用此技术数据。 Loscertales博士的专业知识得到了III Project III投资者Kate Ackerman博士的补充，他在识别小鼠隔膜结构并评估其畸形学方面拥有超过六年的经验。