title: Gene targeting in human-induced pluripotent stem cells with adenoviral vectors.
abstract: Helper-dependent adenoviral vector (HDAdV), which is also called gutless AdV, has been used to deliver donor DNA for gene targeting in human pluripotent stem cells. Surprisingly, the targeting efficacies, both per chromosomal integration (drug-resistant colony) and per treated cells, are much higher than those by standard electroporation and equivalent to those by utilizing artificial nucleases, such as TAL effector nucleases (Aizawa et al., Mol Ther 20:424-431, 2012; Suzuki, Proc Natl Acad Sci U S A 105:13781-13786, 2008). Importantly, gene targeting with HDAdVs was equally efficient in transcriptionally inactive loci in human ES/iPS cells. Therefore, multiple gene-targeted clones can be obtained from human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) cultured in one 100-mm dish. For virus-mediated gene targeting, it is not required to introduce artificial double-strand breaks. By using electroporation for gene targeting, target cells should be expanded to 10⁶-10⁷ cells. In contrast, as an advantage of virus-mediated method, DNA delivery efficiency is high even in a smaller number of cells, resulting in minimizing the number of passages/cell divisions before performing gene targeting. The characteristics suggest that HDAdV-mediated gene targeting has potential advantages for manipulation of chromosomes of pluripotent stem cells for therapeutic applications.
authors.affiliation: Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan.
authors.forename: Kohnosuke
authors.initials: K
authors.lastname: Mitani
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D014161:Transduction, Genetic