Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.
Department of Pediatrics, Amalia Children's Hospital, Nijmegen, The Netherlands.
Methods Mol Biol. 2022;2434:145-165. doi: 10.1007/978-1-0716-2010-6_9.
A significant proportion of mutations underlying genetic disorders affect pre-mRNA splicing, generally causing partial or total skipping of exons, and/or inclusion of pseudoexons. These changes often lead to the formation of aberrant transcripts that can induce nonsense-mediated decay, and a subsequent lack of functional protein. For some genetic disorders, including inherited retinal diseases (IRDs), reproducing splicing dynamics in vitro is a challenge due to the specific environment provided by, e.g. the retinal tissue, cells of which cannot be easily obtained and/or cultured. Here, we describe how to engineer splicing vectors, validate the reliability and reproducibility of alternative cellular systems, assess pre-mRNA splicing defects involved in IRD, and finally correct those by using antisense oligonucleotide-based strategies.
遗传疾病的基因突变中，相当一部分会影响前体 mRNA 的剪接，通常导致外显子部分或全部跳过，和/或假性外显子的包含。这些变化通常会导致形成异常转录本，从而诱导无意义介导的降解，随后导致功能蛋白的缺乏。对于一些遗传疾病，包括遗传性视网膜疾病（IRDs），由于视网膜组织等提供的特定环境，在体外重现剪接动力学是一个挑战，而这些组织中的细胞不容易获得和/或培养。在这里，我们描述了如何设计剪接载体，验证替代细胞系统的可靠性和可重复性，评估与 IRD 相关的前体 mRNA 剪接缺陷，最后通过使用反义寡核苷酸策略来纠正这些缺陷。
