Alternative splicing generates a myriad of Gephyrin isoforms at iPSD. Complex regulation of Gephyrin splicing is a determinant of inhibitory postsynaptic diversity

Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse brain development. We performed a targeted Gphn RNA analysis using Third Generation Sequencing technology developed by Pacific Bioscience (PACBIO). RNA was extracted from mouse cortex and cerebellum tissues as described (Chomczynski P. et al., PMID: 17406285 ), and Reverse Transcription (RT) was performed with the specific primer GPHN-49: GTACTGTGCCTGAGGCTGC. Gphn expression was next amplified using two rounds of PCRs, first 20 cycles with the primers GCAGTCGAACATGTAGCTGACTCAGGTCACCCACGACCATCAAATCCGTGTC and TGGATCACTTGTGCAAGCATCACATCGTAGCAGGATACAGTCAATGATATGTGGACATGC, and after purification of PCR fragment a second PCR of 23 cycles was applied to each sample to add a barcode and allow multiplexing of the library.

gephyrin蛋白（GPHN）可调控抑制性突触的突触后组分聚集，并参与神经精神疾病的病理生理过程。本研究揭示了小鼠脑发育过程中，经剪接严格调控的GPHN转录本存在广泛多样性。本研究采用太平洋生物科学公司（Pacific Bioscience，PACBIO）开发的第三代测序技术（Third Generation Sequencing），开展靶向Gphn RNA分析。按照已发表方法（Chomczynski P.等，PMID: 17406285）从小鼠皮层与小脑组织中提取RNA，并以特异性引物GPHN-49：GTACTGTGCCTGAGGCTGC开展反转录（Reverse Transcription，RT）反应。随后通过两轮聚合酶链式反应（Polymerase Chain Reaction，PCR）扩增Gphn的表达产物：第一轮PCR共20个循环，所用引物为GCAGTCGAACATGTAGCTGACTCAGGTCACCCACGACCATCAAATCCGTGTC与TGGATCACTTGTGCAAGCATCACATCGTAGCAGGATACAGTCAATGATATGTGGACATGC；PCR产物纯化后，对每个样本进行第二轮23个循环的PCR，以添加条形码（barcode）并实现文库的多重测序。