Precise mapping of one classic and three novel GluRIIA mutants in Drosophila melanogaster.
Precise mapping of one classic and three novel GluRIIA mutants in Drosophila melanogaster.
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DOI：
10.17912/micropub.biology.000784
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发表时间：
2023
影响因子：
--
通讯作者：
Frank, C Andrew
中科院分区：
文献类型：
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作者：
Mallik, Bhagaban;Brusich, Douglas J;Heyrman, Georgette;Frank, C Andrew
果蝇GluRIIA基因的突变或靶向该基因的药物常用于评估幼虫神经肌肉接头（NMJ）的稳态突触功能。常用的突变，GluRIIA SP16，是一个无效等位基因产生的一个大的和不精确的切除的P-元件，影响GluRIIA和多个上游基因。在这里，我们绘制了GluRIIA SP16等位基因的确切边界，改进了用于在纯合或杂合背景中阳性鉴定GluRIIA SP16的多重PCR策略，并对三种新的CRISPR产生的GluRIIA突变体进行了测序和表征。我们发现这三个新的GluRIIA等位基因在3龄幼虫NMJ中缺乏GluRIIA免疫荧光信号，并被预测在遗传水平上导致过早截短。此外，这些新的突变体具有与GluRIIA SP16类似的电生理结果，包括与对照相比降低的微型兴奋性突触后电位（mEPSP）幅度和频率，并且它们表达稳健的稳态补偿，如由正常兴奋性突触后电位（EPSP）幅度和升高的量子含量所证明的。这些发现和新工具扩展了D.用于评估突触功能的黑腹NMJ。
Mutation of the Drosophila melanogaster GluRIIA gene or pharmacological agents targeting it are commonly used to assess homeostatic synaptic function at the larval neuromuscular junction (NMJ). The commonly used mutation, GluRIIA SP16 , is a null allele created by a large and imprecise excision of a P-element which affects GluRIIA and multiple upstream genes. Here we mapped the exact bounds of the GluRIIA SP16 allele, refined a multiplex PCR strategy for positive identification of GluRIIA SP16 in homozygous or heterozygous backgrounds, and sequenced and characterized three new CRISPR-generated GluRIIA mutants. We found the three new GluRIIA alleles are apparent nulls that lack GluRIIA immunofluorescence signal at the 3 rd instar larval NMJ and are predicted to cause premature truncations at the genetic level. Further, these new mutants have similar electrophysiological outcomes as GluRIIA SP16 , including reduced miniature excitatory postsynaptic potential (mEPSP) amplitude and frequency compared to controls, and they express robust homeostatic compensation as evidenced by normal excitatory postsynaptic potential (EPSP) amplitude and elevated quantal content. These findings and new tools extend the capacity of the D. melanogaster NMJ for assessment of synaptic function.