Desviat Lourdes R, Pérez Belén, Ugarte Magdalena
Centro de Diagnóstico de Enfermedades Moleculares, Madrid, Spain.
Methods Mol Biol. 2012;867:37-47. doi: 10.1007/978-1-61779-767-5_3.
Although several bioinformatic tools exist to predict the effect on splicing of a nucleotide change, experimental verification with minigenes is essential for diagnostic purposes, as well as for revealing disease mechanisms and monitoring therapeutic interventions. Minigenes are splice reporter vectors (also known as exon-trapping vectors) that allow confirmation of the effect of mutations on the splicing process, indicated when patients' samples for RNA studies are not available. The minigene vector codes for exonic portions of a gene defined by functional 5' splice donor and 3' splice acceptor sites separated by intronic sequences where a polylinker is located. Here, the exon carrying the mutation under study is cloned along with its flanking intronic sequence. The resulting construct, in its wild-type and mutant sequence version, is transfected in established cell lines and the vector splicing pattern is analyzed. Ideally, the wild-type minigene results in correct exon inclusion, while the mutant construct results in exon skipping or other aberrant transcripts.
虽然有几种生物信息学工具可用于预测核苷酸变化对剪接的影响，但为了诊断目的、揭示疾病机制以及监测治疗干预，使用微型基因进行实验验证至关重要。微型基因是剪接报告载体（也称为外显子捕获载体），当无法获得用于RNA研究的患者样本时，它可以确认突变对剪接过程的影响。微型基因载体编码由功能性5'剪接供体和3'剪接受体位点定义的基因外显子部分，这些位点由内含子序列分隔，内含子序列中含有一个多克隆位点。在此，将携带正在研究的突变的外显子与其侧翼内含子序列一起克隆。将所得构建体的野生型和突变序列版本转染到已建立的细胞系中，并分析载体的剪接模式。理想情况下，野生型微型基因会导致正确的外显子包含，而突变构建体会导致外显子跳跃或其他异常转录本。
