Hasegawa Yoshinori, Ikeno Masashi, Suzuki Nobutaka, Nakayama Manabu, Ohara Osamu
Laboratory of Clinical Omics Research, Department of Applied Genomics, Kazusa DNA Research Institute, Chiba, Japan.
Aichi Medical University, Aichi, Japan.
Biol Methods Protoc. 2018 Dec 31;3(1):bpy013. doi: 10.1093/biomethods/bpy013. eCollection 2018.
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.
人类人工染色体（HAC）载体有潜力克服与基于质粒、转座子和病毒的载体相关的稳定基因表达问题，如插入诱变、位置效应、不可控的拷贝数、不稳定的基因表达以及DNA大小限制。HAC的主要优点是其游离性质和容纳任何大小DNA插入片段的能力。然而，HAC载体有两个缺点：基因插入效率低以及缺乏关于成功将HAC转移到人类诱导多能干细胞（iPSC）的报道。我们在此首次报道了一种将HAC高效转移到人类iPSC以获得可重复实验结果的方法。此外，使用我们新的位点特异性重组系统VCre/VloxP和SCre/SloxP，我们使HAC载体中的基因插入效率提高了10%。
