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

• 批准号: 8051050
• 负责人: JAMES H. MILLONIG
• 金额: $ 22.71万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-05 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8051050
• 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; 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; recombinase; red fluorescent protein; research study; transcription factor; vector

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）是一种常见的衰弱性神经发育障碍。尽管有强有力的证据表明遗传因素对自闭症谱系障碍有影响，但分离特定的致病遗传缺陷一直很困难。我们之前的研究主要集中在同源盒转录因子ENGRAILED 2 （EN2）上。我们已经在三个独立的数据集中证明了两个内含子EN2 snp rs1861972和rs1861973的一致性关联。这些发现表明EN2很可能是一种ASD易感基因。LD定位和重测序鉴定出相关的a - c rs1861972-rs1816973单倍型作为候选疾病等位基因。功能研究表明，EN2内含子是一种转录抑制因子，而a - c单倍型与不相关的G-T单倍型相比，产生较弱的抑制因子。emsa已经确定DNA结合蛋白特异性地与两个snp的相关等位基因相互作用，为观察到的功能差异提供了一种机制。这些研究已经确定了A-C单倍型是自闭症的第一个常见风险因素。本研究的目的是利用重组酶介导的基因组替代（RMGR）技术建立EN2风险等位基因的小鼠模型。RMGR比标准靶向方法更可取，因为小鼠基因组的大片段（bbb100kb）可以被合成的人类区域取代。这减少了对人类基因适当调控的担忧。我们的计划是使用RMGR将约72kb的小鼠En2基因座替换为人类的同源区域，并为相关的A-C和非相关的G-T单倍型生成敲入系。一个IRES：红色荧光蛋白（DsRed-E5/pTIMER）也将用于修饰EN2位点，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。然后对敲入基因进行检查，以确定EN2风险等位基因发挥功能的发育细胞类型和年龄。对En2基因敲除的研究揭示了与ASD相关的解剖学、发育和神经化学表型。作为确定受风险等位基因影响的细胞途径的第一步，将对敲入基因重复进行相同的分析。这一信息对于更好地诊断、治疗和预防自闭症谱系障碍至关重要。