Search

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 MYH7genes. These results indicate that the double-tk donor vector system enables efficient selection of knocked-in hiPSCs carrying reporter proteins.

Survivors of pediatric brain tumors are often affected by late effects, such as motility disturbance of limb(s), seizure, ocularvisual impairment, endocrine abnormality, and higher brain dysfunction, resulting from the disease and its treatment. Appropriate provision of supportive care will require understanding the effects of these experiences on survivors’ health-related quality of life (HRQOL).