A versatile toolbox for knock-in gene targeting based on the Multisite Gateway technology
A versatile toolbox for knock-in gene targeting based on the Multisite Gateway technology
复制标题
DOI：
10.1371/journal.pone.0221164
复制
发表时间：
2019-08
期刊：
影响因子：
3.7
通讯作者：
Sho Yoshimatsu;T. Sone;Mayutaka Nakajima;Tsukika Sato;Ryotaro Okochi;Mitsuru Ishikawa;Mari Nakamura;E. Sasaki;Seiji Shiozawa;H. Okano
中科院分区：
文献类型：
--
作者：
Sho Yoshimatsu;T. Sone;Mayutaka Nakajima;Tsukika Sato;Ryotaro Okochi;Mitsuru Ishikawa;Mari Nakamura;E. Sasaki;Seiji Shiozawa;H. Okano
敲入(KI)基因打靶在干细胞研究中具有广泛的应用前景。然而，KI基因载体的构建需要多个复杂的过程，包括多次的消化/连接步骤和广泛的限制性内切酶图谱，这限制了KI基因打靶的广泛应用。为了规避这个问题，我们在这里介绍了通过分子克隆产生KI载体的通用和系统的方法。在这种方法中，我们使用了多站点网关技术，这是一种使用专利序列和酶的高效体外DNA重组系统。利用这些方法构建KI载体只需要有效的步骤，如聚合酶链式反应和重组，从而实现强大的KI基因靶向。我们发现，使用这种方法产生的KI载体和位点特异性核酸酶的组合使用，使得荧光蛋白基因在人类和常见的绒猴(绒猴；Callithrix Jacchus)多个基因座的多个位点上能够精确整合。这里描述的方法将促进KI技术的使用，并最终有助于加快干细胞研究。
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.