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

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.