Generation of targeted homozygosity in the genome of human induced pluripotent stem cells


Autoři: Yasuhide Yoshimura aff001;  Ayako Yamanishi aff001;  Tomo Kamitani aff001;  Jin-Soo Kim aff002;  Junji Takeda aff001
Působiště autorů: Department of Genome Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan aff001;  Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0225740

Souhrn

When loss of heterozygosity (LOH) is correlated with loss or gain of a disease phenotype, it is often necessary to identify which gene or genes are involved. Here, we developed a region-specific LOH-inducing system based on mitotic crossover in human induced pluripotent stem cells (hiPSCs). We first tested our system on chromosome 19. To detect homozygous clones generated by LOH, a positive selection cassette was inserted at the AASV1 locus of chromosome 19. LOHs were generated by the combination of allele-specific double-stranded DNA breaks introduced by CRISPR/Cas9 and suppression of Bloom syndrome (BLM) gene expression by the Tet-Off system. The BLM protein inhibitor ML216 exhibited a similar crossover efficiency and distribution of crossover sites. We next applied this system to the short arm of chromosome 6, where human leukocyte antigen (HLA) loci are located. Genotyping and flow cytometric analysis demonstrated that LOHs associated with chromosomal crossover occurred at the expected positions. Although careful examination of HLA-homozygous hiPSCs generated from parental cells is needed for cancer predisposition and effectiveness of differentiation, they may help to mitigate the current shortcoming of hiPSC-based transplantation related to the immunological differences between the donor and host.

Klíčová slova:

Genetic loci – Homozygosity – Mammalian genomics – Molecular genetics – Polymerase chain reaction – Transfection – Electroporation


Zdroje

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Článok vyšiel v časopise

PLOS One


2019 Číslo 12