An Adapted GeneSwitch Toolkit for Comparable Cellular and Animal Models: A Proof of Concept in Modeling Charcot-Marie-Tooth Neuropathy.
An Adapted GeneSwitch Toolkit for Comparable Cellular and Animal Models: A Proof of Concept in Modeling Charcot-Marie-Tooth Neuropathy.
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DOI：
10.3390/ijms242216138
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发表时间：
2023-11-09
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
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使用疾病建模研究致病突变的影响、其受影响的途径和/或潜在的治疗策略通常需要生成不同的体内和细胞内模型。迄今为止，已经建立了几种方法来在不同的模型系统中以受控的方式诱导转基因表达。然而，根据所使用的模式生物，需要进行几轮亚克隆，从而将劳动密集型实验纳入技术方法和分析比较。GeneSwitch ™技术是经典UAS-GAL4诱导型系统的改良版本，允许转基因表达的空间和时间调节。它由三个组分组成：编码嵌合调节pSwitch蛋白的质粒、作为诱导剂的米非司酮和诱导型质粒。虽然含有pSwitch的第一质粒可以在体内和细胞内使用，但诱导型第二质粒只能在细胞内使用。这需要针对所使用的模式生物定制的诱导型质粒的特定亚克隆策略。为了避免这一步骤并统一所产生的转基因模型中的基因表达，对于含有嵌合GeneSwitch ™ cDNA序列或转基因cDNA序列的两种质粒，我们用标准pUAS-attB质粒替换了骨架载体。我们优化了这种适应系统，以调节几种哺乳动物细胞系中的转基因表达。此外，我们利用这个新系统来生成YARS 1诱导的Charco-Marie-Tooth神经病（CMT）的统一细胞和果蝇模型。这些新模型显示了预期的CMT样表型。在表达YARS1转基因的N2a神经母细胞瘤细胞中，我们观察到合成酶的典型"泪滴"分布，该分布在表达YARS1CMT突变时受到干扰。在果蝇中，YARS1CMT的普遍表达诱导剂量依赖性发育致死性和泛神经元表达引起运动缺陷，而野生型等位基因的表达是无害的。我们的概念验证疾病建模研究支持适应性转基因系统作为一种强大工具的功效，允许设计具有最佳数据可比性的研究。
Investigating the impact of disease-causing mutations, their affected pathways, and/or potential therapeutic strategies using disease modeling often requires the generation of different in vivo and in cellulo models. To date, several approaches have been established to induce transgene expression in a controlled manner in different model systems. Several rounds of subcloning are, however, required, depending on the model organism used, thus bringing labor-intensive experiments into the technical approach and analysis comparison. The GeneSwitch™ technology is an adapted version of the classical UAS-GAL4 inducible system, allowing the spatial and temporal modulation of transgene expression. It consists of three components: a plasmid encoding for the chimeric regulatory pSwitch protein, Mifepristone as an inducer, and an inducible plasmid. While the pSwitch-containing first plasmid can be used both in vivo and in cellulo, the inducible second plasmid can only be used in cellulo. This requires a specific subcloning strategy of the inducible plasmid tailored to the model organism used. To avoid this step and unify gene expression in the transgenic models generated, we replaced the backbone vector with standard pUAS-attB plasmid for both plasmids containing either the chimeric GeneSwitch™ cDNA sequence or the transgene cDNA sequence. We optimized this adapted system to regulate transgene expression in several mammalian cell lines. Moreover, we took advantage of this new system to generate unified cellular and fruit fly models for YARS1-induced Charco–Marie–Tooth neuropathy (CMT). These new models displayed the expected CMT-like phenotypes. In the N2a neuroblastoma cells expressing YARS1 transgenes, we observed the typical “teardrop” distribution of the synthetase that was perturbed when expressing the YARS1CMT mutation. In flies, the ubiquitous expression of YARS1CMT induced dose-dependent developmental lethality and pan-neuronal expression caused locomotor deficit, while expression of the wild-type allele was harmless. Our proof-of-concept disease modeling studies support the efficacy of the adapted transgenesis system as a powerful tool allowing the design of studies with optimal data comparability.
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影响因子：
16.6
作者：
Bervoets, Sven;Wei, Na;Yang, Xiang-Lei
通讯作者：
Yang, Xiang-Lei
DOI：
10.1073/pnas.90.5.1657
发表时间：
1993-03-01
影响因子：
11.1
作者：
BRASELMANN, S;GRANINGER, P;BUSSLINGER, M
通讯作者：
BUSSLINGER, M
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
9.8
作者：
Antonellis, A;Ellsworth, RE;Green, ED
通讯作者：
Green, ED
影响因子：
3.7
作者：
Ahler E;Sullivan WJ;Cass A;Braas D;York AG;Bensinger SJ;Graeber TG;Christofk HR
通讯作者：
Christofk HR