Improving the efficiency of gene insertion in a human artificial chromosome vector and its transfer in human-induced pluripotent stem cells

Yoshinori Hasegawa; Masashi Ikeno; Nobutaka Suzuki; Manabu Nakayama; Osamu Ohara

doi:10.1093/biomethods/bpy013

Improving the efficiency of gene insertion in a human artificial chromosome vector and its transfer in human-induced pluripotent stem cells

Biol Methods Protoc. 2018 Dec 31;3(1):bpy013. doi: 10.1093/biomethods/bpy013. eCollection 2018.

Authors

Yoshinori Hasegawa¹, Masashi Ikeno², Nobutaka Suzuki³, Manabu Nakayama⁴, Osamu Ohara¹

Affiliations

¹ Laboratory of Clinical Omics Research, Department of Applied Genomics, Kazusa DNA Research Institute, Chiba, Japan.
² Aichi Medical University, Aichi, Japan.
³ Chromoresearch Inc., Aichi, Japan.
⁴ Laboratory of Medical Omics Research, Department of Frontier Research and Development, Kazusa DNA Research Institute, Chiba, Japan.

Abstract

A human artificial chromosome (HAC) vector has potential to overcome the problems of stable gene expression associated with plasmid, transposon, and virus-based vectors, such as insertional mutagenesis, position effect, uncontrollable copy number, unstable gene expression, and DNA size limitation. The main advantages of the HAC are its episomal nature and ability to accommodate DNA inserts of any size. However, HAC vectors have two disadvantages: low efficiency of gene insertion and lack of reports regarding the successful HAC transfer to human-induced pluripotent stem cells (iPSCs). We here provide the first report of a method for the efficient transfer of HAC to human iPSCs for obtaining reproducible experimental results. Moreover, we achieved a 10% increase in the gene insertion efficiency in the HAC vector using our new site-specific recombination systems VCre/VloxP and SCre/SloxP.

Keywords: Human artificial chromosome (HAC) vector; dual RMCE; hiPSC.