Mallik Bhagaban, Brusich Douglas J, Heyrman Georgette, Frank C Andrew
Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa, United States.
Human Biology Department, University of Wisconsin-Green Bay, Green Bay, Wisconsin, United States.
MicroPubl Biol. 2023 Jun 2;2023. doi: 10.17912/micropub.biology.000784. eCollection 2023.
Mutation of the gene or pharmacological agents targeting it are commonly used to assess homeostatic synaptic function at the larval neuromuscular junction (NMJ). The commonly used mutation, , is a null allele created by a large and imprecise excision of a P-element which affects and multiple upstream genes. Here we mapped the exact bounds of the allele, refined a multiplex PCR strategy for positive identification of in homozygous or heterozygous backgrounds, and sequenced and characterized three new CRISPR-generated mutants. We found the three new alleles are apparent nulls that lack GluRIIA immunofluorescence signal at the 3 instar larval NMJ and are predicted to cause premature truncations at the genetic level. Further, these new mutants have similar electrophysiological outcomes as , 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 NMJ for assessment of synaptic function.
针对该基因的突变或靶向它的药物制剂通常用于评估幼虫神经肌肉接头（NMJ）处的稳态突触功能。常用的突变体 是通过P元件的大而不精确切除产生的无效等位基因，它影响 以及多个上游基因。在这里，我们绘制了 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 的多重PCR策略，并对三个新的CRISPR产生的 突变体进行了测序和表征。我们发现这三个新的 等位基因明显是无效的，在三龄幼虫NMJ处缺乏GluRIIA免疫荧光信号，并且预计在基因水平上会导致过早截断。此外，这些新突变体具有与 相似的电生理结果，包括与对照相比，微小兴奋性突触后电位（mEPSP）幅度和频率降低，并且它们表现出强大的稳态补偿，正常兴奋性突触后电位（EPSP）幅度和升高的量子含量证明了这一点。这些发现和新工具扩展了NMJ评估突触功能的能力。
