A Mouse Knock-In Model for ENGRAILED 2 Autism Susceptibility

ENGRAILED 2 自闭症易感性的小鼠敲入模型

基本信息

批准号：
7491964
负责人：
JAMES H. MILLONIG
金额：
$ 15.28万
依托单位：
UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-05-05 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7491964
关键词：
A Mouse ARHGEF5 gene Affect Age Alleles Autistic Disorder Binding Biochemical Markers Biological Biological Assay Cell Cycle Cell Line Cerebellum Chimera organism Code Collaborations DNA DNA-Binding Proteins Data Data Set Defect Development Diagnosis Disease DsRed Embryo Engineering Exons Family Gene Expression Regulation Generations Genes Genetic Genomics Goals Haplotypes High Pressure Liquid Chromatography Histological Techniques Homeobox Human In Vitro Individual Introns Knock-in Mouse Knock-out Luciferases Maps Mediating Modeling Modification Mus Mutant Strains Mice Mutation National Institute of Mental Health Neurodevelopmental Disorder Neuroglia Neurons Neurotransmitters Norepinephrine Pathway interactions Pattern Phase Phenotype Population Population Attributable Risks Predisposition Principal Investigator Procedures Proteins Protocols documentation Publishing Regulation Reporter Reporting Research Risk Risk Factors Sampling Screening procedure Serotonin Site Standards of Weights and Measures Students Susceptibility Gene Tail Techniques Testing Time Transcription Repressor/Corepressor Transgenic Organisms Universities Variant autism spectrum disorder cell type disorder risk embryonic stem cell genetic risk factor granule cell homologous recombination improved in vivo mouse genome mouse model neurochemistry programs recombinase red fluorescent protein research study transcription factor vector

项目摘要

DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) is a common and debilitating neurodevelopmental disorder. Although there is strong evidence for a genetic contribution to ASD, the isolation of specific causative genetic defects has been difficult. Our previous research has focused on the homeobox transcription factor ENGRAILED 2 (EN2). We have demonstrated consistent association for two intronic EN2 SNPs, rs1861972 and rs1861973, in three separate datasets. These findings determined that EN2 is a likely ASD susceptibility gene. LD mapping and re-sequencing identified the associated A-C rs1861972-rs1816973 haplotype as a candidate disease allele. Functional studies have now demonstrated that the EN2 intron acts a transcriptional repressor and that the A-C haplotype results in a weaker repressor compared to the non-associated G-T haplotype. EMSAs have determined that DNA binding proteins specifically interact with the associated alleles for both SNPs, providing a mechanism for the observed functional difference. These studies have identified the A-C haplotype as the first common risk factor for autism. The goal of this proposal is to generate a mouse model for the EN2 risk allele by using recombinase- mediated genomic replacement (RMGR). RMGR is preferred over standard targeting approaches because large segments of the mouse genome (>100kb) can be replaced with the syntenic human region. This reduces concerns about the proper regulation of the human gene. Our plan is to use RMGR to replace ~72kb of the mouse En2 locus with the syntenic human region and to generate knock-in lines for both the associated A-C and the non-associated G-T haplotypes. An IRES:red fluorescent protein (DsRed-E5/pTIMER) will also be used to modify the EN2 locus so that subtle effects of the risk allele on levels and spatial/temporal expression can be identified. The knock-ins will then be examined to determine the developmental cell types and ages in which the EN2 risk allele is functional. Studies with the En2 knock-out have uncovered anatomical, developmental and neurochemical phenotypes relevant to ASD. The same analysis will be repeated for the knock-ins as a first step in determining the cellular pathways affected by the risk allele. This information will be critical in the development of better diagnoses, treatments and preventions for ASD.

描述（由申请人提供）：自闭症谱系障碍（ASD）是一种常见且令人衰弱的神经发育障碍。尽管有强有力的证据表明对ASD有遗传贡献，但很难分离特定的病因遗传缺陷。我们以前的研究重点是植入2（EN2）的同型转录因子。在三个单独的数据集中，我们已经证明了两个内含子EN2 SNP RS1861972和RS1861973的关联。这些发现确定EN2可能是ASD敏感性基因。 LD映射和重新测序确定了相关的A-C RS1861972-RS1816973单倍型为候选等位基因。现在，功能研究表明，与非相关的G-T单倍型相比，EN2内含子起作用转录阻遏物，A-C单倍型会导致较弱的阻遏物。 EMSAS已确定DNA结合蛋白与两个SNP的相关等位基因特别相互作用，从而为观察到的功能差异提供了一种机制。这些研究将A-C单倍型确定为自闭症的第一个常见危险因素。该提案的目的是通过使用重组酶介导的基因组替代（RMGR）生成EN2风险等位基因的小鼠模型。与标准靶向方法相比，RMGR优选，因为可以用同义人类区域代替小鼠基因组（> 100KB）的大段。这减少了对人类基因适当调节的担忧。我们的计划是使用RMGR用同步人类区域代替〜72KB的小鼠EN2基因座，并为相关的A-C和非相关的G-T单倍型生成敲门线。 IRES：红色荧光蛋白（DSRED-E5/PTIMER）也将用于修饰EN2基因座，以便可以鉴定出风险等位基因对水平和空间/时间表达的微妙影响。然后，将检查敲门剂，以确定EN2风险等位基因功能性的发育细胞类型和年龄。通过EN2敲除的研究发现了与ASD相关的解剖，发育和神经化学表型。对于确定受风险等位基因影响的细胞途径的第一步，将重复相同的分析。此信息对于ASD的更好诊断，治疗和预防措施的发展至关重要。