Emily Brown, Samantha Kuszynski, Faith Akoachere, James Feduccia, Lili Malatinszky, Eric S Luth
{"title":"生成内源性辅酶诱导性脱粒子标记的 SPAS-1/spastin，以研究其在秀丽隐杆线虫神经元中的定向消耗。","authors":"Emily Brown, Samantha Kuszynski, Faith Akoachere, James Feduccia, Lili Malatinszky, Eric S Luth","doi":"10.17912/micropub.biology.001328","DOIUrl":null,"url":null,"abstract":"<p><p>To facilitate investigations of the microtubule severing protein spastin and its specific role in neurons, we aimed to create a <i>C. elegans</i> strain in which the spastin homolog SPAS-1 is visible and can be degraded with spatial and temporal precision. We used CRISPR-Cas9 to fuse an auxin-inducible degron and mScarlet to the endogenous SPAS-1 protein, enabling degradation of SPAS-1 in neurons during desired life stages. DNA sequencing confirmed in-frame insertion with the SPAS-1 N-terminus and fluorescence microscopy revealed endogenous SPAS-1 throughout the CRISPR-edited worms. Auxin treatment in <i>rgef-1::TIR1; mScarlet::AID*::3xFLAG::spas-1</i> animals reduced mScarlet::SPAS-1 fluorescence in neuronal ganglia.</p>","PeriodicalId":74192,"journal":{"name":"microPublication biology","volume":"2024 ","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11582882/pdf/","citationCount":"0","resultStr":"{\"title\":\"Generation of an endogenous auxin inducible degron-tagged SPAS-1/spastin to investigate its targeted depletion in <i>C. elegans</i> neurons.\",\"authors\":\"Emily Brown, Samantha Kuszynski, Faith Akoachere, James Feduccia, Lili Malatinszky, Eric S Luth\",\"doi\":\"10.17912/micropub.biology.001328\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>To facilitate investigations of the microtubule severing protein spastin and its specific role in neurons, we aimed to create a <i>C. elegans</i> strain in which the spastin homolog SPAS-1 is visible and can be degraded with spatial and temporal precision. We used CRISPR-Cas9 to fuse an auxin-inducible degron and mScarlet to the endogenous SPAS-1 protein, enabling degradation of SPAS-1 in neurons during desired life stages. DNA sequencing confirmed in-frame insertion with the SPAS-1 N-terminus and fluorescence microscopy revealed endogenous SPAS-1 throughout the CRISPR-edited worms. Auxin treatment in <i>rgef-1::TIR1; mScarlet::AID*::3xFLAG::spas-1</i> animals reduced mScarlet::SPAS-1 fluorescence in neuronal ganglia.</p>\",\"PeriodicalId\":74192,\"journal\":{\"name\":\"microPublication biology\",\"volume\":\"2024 \",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-11-07\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11582882/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"microPublication biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.17912/micropub.biology.001328\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"microPublication biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.17912/micropub.biology.001328","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"","JCRName":"","Score":null,"Total":0}
Generation of an endogenous auxin inducible degron-tagged SPAS-1/spastin to investigate its targeted depletion in C. elegans neurons.
To facilitate investigations of the microtubule severing protein spastin and its specific role in neurons, we aimed to create a C. elegans strain in which the spastin homolog SPAS-1 is visible and can be degraded with spatial and temporal precision. We used CRISPR-Cas9 to fuse an auxin-inducible degron and mScarlet to the endogenous SPAS-1 protein, enabling degradation of SPAS-1 in neurons during desired life stages. DNA sequencing confirmed in-frame insertion with the SPAS-1 N-terminus and fluorescence microscopy revealed endogenous SPAS-1 throughout the CRISPR-edited worms. Auxin treatment in rgef-1::TIR1; mScarlet::AID*::3xFLAG::spas-1 animals reduced mScarlet::SPAS-1 fluorescence in neuronal ganglia.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
