Open AccessA versatile toolbox for knock-in gene targeting based on the Multisite Gateway technology
Author(s) -
Sho Yoshimatsu,
Takefumi Sone,
Mayutaka Nakajima,
Takao Sato,
Ryotaro Okochi,
Mitsuru Ishikawa,
Mari Nakamura,
Erika Sasaki,
Sho Shiozawa,
Hideyuki Okano
Publication year - 2019
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0221164
Subject(s) - computational biology , biology , induced pluripotent stem cell , restriction enzyme , crispr , gene , restriction digest , multiple cloning site , gene targeting , computer science , genetics , vector (molecular biology) , embryonic stem cell , recombinant dna
Knock-in (KI) gene targeting can be employed for a wide range of applications in stem cell research. However, vectors for KI require multiple complicated processes for construction, including multiple times of digestion/ligation steps and extensive restriction mapping, which has imposed limitations for the robust applicability of KI gene targeting. To circumvent this issue, here we introduce versatile and systematic methods for generating KI vectors by molecular cloning. In this approach, we employed the Multisite Gateway technology, an efficient in vitro DNA recombination system using proprietary sequences and enzymes. KI vector construction exploiting these methods requires only efficient steps, such as PCR and recombination, enabling robust KI gene targeting. We show that combinatorial usage of the KI vectors generated using this method and site-specific nucleases enabled the precise integration of fluorescent protein genes in multiple loci of human and common marmoset (marmoset; Callithrix jacchus ) pluripotent stem cells. The methods described here will facilitate the usage of KI technology and ultimately help to accelerate stem cell research.