Tidball Andrew M, Swaminathan Preethi, Dang Louis T, Parent Jack M
Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, USA.
Department of Pediatrics, University of Michigan Medical School, Ann Arbor, MI, USA.
Bio Protoc. 2018 Apr 5;8(7). doi: 10.21769/BioProtoc.2794.
For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones.
对于疾病研究和基础科学研究而言，功能丧失（LOF）突变都至关重要。在此，我们提供了一种简单的简化方案，用于生成LOF诱导多能干细胞（iPSC）系，该方案通过将CRISPR载体与游离重编程质粒同时导入成纤维细胞，规避了传统基因编辑和已建立的iPSC系克隆的技术挑战。我们的实验在常染色体基因中产生了几乎数量均等的所有3种基因型。此外，我们还提供了一种详细的方法，用于维持iPSC克隆的96孔板并进行基因分型。
