Multiple expression cassette exchange via TP901-1, R4, and Bxb1 integrase systems on a mouse artificial chromosome.
Multiple expression cassette exchange via TP901-1, R4, and Bxb1 integrase systems on a mouse artificial chromosome.
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DOI：
10.1002/2211-5463.12169
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发表时间：
2017-03
期刊：
影响因子：
2.6
通讯作者：
Ohbayashi T
中科院分区：
文献类型：
--
作者：
Tomimatsu K;Kokura K;Nishida T;Yoshimura Y;Kazuki Y;Narita M;Oshimura M;Ohbayashi T
基因敲除或谱系追踪是研究生物系统的有力工具。目前，位点特异性重组酶（SSRs），如Cre或Flp重组靶盒，已经成功地通过单个SSR切除或倒置来调节转基因表达。然而，单一SSR的使用可能会限制基因表达的复杂控制。本研究探讨了使用三种不同的整合酶系统（TP901‐1、R4和Bxb1）扩大转基因多重调控的潜力。我们设计了三个表达荧光素酶的切除盒，其中荧光素酶的表达可以通过位点特异性重组交换为荧光蛋白。将可被不同整合酶独立调控的单个磁带串联并插入到中国仓鼠卵巢细胞的小鼠人工染色体（MAC）载体中。整合酶的瞬时表达导致目标荧光素酶活性丧失，荧光被激活。此外，整合酶系统能够在不与其他重组靶标发生交叉反应的情况下特异性切除目标DNA序列。这些结果表明，这些整合酶系统在MAC载体上的特定位点上的联合使用允许转基因表达的多重调控，并可能有助于基因组或细胞工程。
The site‐specific excision of a target DNA sequence for genetic knockout or lineage tracing is a powerful tool for investigating biological systems. Currently, site‐specific recombinases (SSRs), such as Cre or Flp recombination target cassettes, have been successfully excised or inverted by a single SSR to regulate transgene expression. However, the use of a single SSR might restrict the complex control of gene expression. This study investigated the potential for expanding the multiple regulation of transgenes using three different integrase systems (TP901‐1, R4, and Bxb1). We designed three excision cassettes that expressed luciferase, where the luciferase expression could be exchanged to a fluorescent protein by site‐specific recombination. Individual cassettes that could be regulated independently by a different integrase were connected in tandem and inserted into a mouse artificial chromosome (MAC) vector in Chinese hamster ovary cells. The transient expression of an integrase caused the targeted luciferase activity to be lost and fluorescence was activated. Additionally, the integrase system enabled the specific excision of targeted DNA sequences without cross‐reaction with the other recombination targets. These results suggest that the combined use of these integrase systems in a defined locus on a MAC vector permits the multiple regulation of transgene expression and might contribute to genomic or cell engineering.