整合酶
整合酶
突变体
生物
中国仓鼠卵巢细胞
遗传学
点突变
转基因
基因座(遗传学)
基因
计算生物学
细胞培养
作者
Barbara Jusiak,Kalpana Jagtap,Leonid Gaidukov,Xavier Duportet,Kalpanie Bandara,Jianlin Chu,Lin Zhang,Ron Weiss,Timothy K. Lu
标识
DOI:10.1021/acssynbio.8b00089
摘要
Phage-derived integrases can catalyze irreversible, site-specific integration of transgenic payloads into a chromosomal locus, resulting in mammalian cells that stably express transgenes or circuits of interest. Previous studies have demonstrated high-efficiency integration by the Bxb1 integrase in mammalian cells. Here, we show that a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in Chinese hamster ovary cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells. Bxb1-GA point mutant sites do not cross-react with Bxb1 wild-type sites, enabling their use in applications that require orthogonal pairs of target sites. In comparison, we test the efficiency and orthogonality of ϕC31 and Wβ integrases, and show that Wβ has an integration efficiency between those of Bxb1-GA and wild-type Bxb1. Our data present a toolbox of integrases for inserting payloads such as gene circuits or therapeutic transgenes into mammalian cell lines.
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