We modify iPSC lines deposited in and provided by RIKEN cell bank in order to enhance their usefulness. The type and methods of the modification are as follows:
- We generate isogenic control cells by correcting specific mutations responsible for a disease using genome editing technology.
- We generate mutation-introduced iPSC lines if the responsible genes are identified, but the number of disease-specific iPSC lines is not enough to be examined. We use healthy-donor iPSC lines provided by the RIKEN cell bank as the original iPSC lines for this purpose.
- We generate reporter-introduced iPSC lines from disease-specific or healthy-donor iPSC lined with in order to monitor the differentiation status visually by using transgenic or knock-in to tissue or cell type specific promoters with fluorescent proteins.
Generation of two ISL1-tdTomato reporter human induced pluripotent stem cell lines using CRISPR-Cas9 genome editing
ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.
(Tsukamoto et al., Stem Cell Research 2021)