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：
7619517
负责人：
JAMES H. MILLONIG
金额：
$ 15.27万
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/7619517
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 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）是一种常见且使人衰弱的神经发育障碍。尽管有强有力的证据表明遗传因素对自闭症谱系障碍有一定影响，但分离出特定的致病基因缺陷仍然很困难。我们之前的研究重点是同源盒转录因子 ENGRAILED 2 (EN2)。我们在三个独立的数据集中证明了两个内含子 EN2 SNP rs1861972 和 rs1861973 的一致关联。这些发现确定 EN2 可能是 ASD 易感基因。 LD 作图和重测序将相关的 A-C rs1861972-rs1816973 单倍型鉴定为候选疾病等位基因。功能研究现已证明 EN2 内含子起到转录抑制因子的作用，并且与非相关 G-T 单倍型相比，A-C 单倍型产生的抑制因子较弱。 EMSA 已确定 DNA 结合蛋白与两个 SNP 的相关等位基因特异性相互作用，为观察到的功能差异提供了机制。这些研究已将 A-C 单倍型确定为自闭症的第一个常见危险因素。该提案的目标是通过使用重组酶介导的基因组替换 (RMGR) 生成 EN2 风险等位基因的小鼠模型。 RMGR 优于标准靶向方法，因为小鼠基因组的大片段 (>100kb) 可以被同线性人类区域替换。这减少了对人类基因正确调节的担忧。我们的计划是使用 RMGR 将约 72kb 的小鼠 En2 基因座替换为同线性人类区域，并生成相关 A-C 和非相关 G-T 单倍型的敲入系。 IRES:红色荧光蛋白 (DsRed-E5/pTIMER) 还将用于修饰 EN2 基因座，以便识别风险等位基因对水平和空间/时间表达的微妙影响。然后将检查敲入以确定 EN2 风险等位基因发挥功能的发育细胞类型和年龄。 En2 敲除研究揭示了与 ASD 相关的解剖学、发育和神经化学表型。将对敲入重复相同的分析，作为确定受风险等位基因影响的细胞途径的第一步。这些信息对于开发更好的 ASD 诊断、治疗和预防至关重要。