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.
Efficient selection of knocked-in pluripotent stem cells using a dual cassette cellular elimination system
Although recent advances in genome editing technology with homology-directed repair have enabled the insertion of various reporter genes into the genome of mammalian cells, the efficiency is still low due to the random insertion of donor vectors into the host genome. To efficiently select knocked-in cells without random insertion, we developed the “double-tk donor vector system,” in which the expression units of the thymidine kinase of herpes simplex virus (HSV-tk) are placed on both outer sides of homology arms. This system is superior in enriching knocked-in human induced pluripotent stem cells (hiPSCs) than conventional donor vector systems with a single or no HSV-tk cassette. Using this system, we efficiently generated fluorescent reporter knockin hiPSCs targeting POU5F1 (OCT3/4), EEF1A1, H2BC21 (H2B clustered histone 21), ISL1, and MYH7 genes. These results indicate that the double-tk donor vector system enables efficient selection of knocked-in hiPSCs carrying reporter proteins.
(Nakade et al., Cell Reports Methods 2023)