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通过TP901-1、R4和Bxb1整合酶系统在小鼠人工染色体上进行多表达盒交换

Multiple expression cassette exchange via TP901-1, R4, and Bxb1 integrase systems on a mouse artificial chromosome.

作者信息

Tomimatsu Kosuke, Kokura Kenji, Nishida Tadashi, Yoshimura Yuki, Kazuki Yasuhiro, Narita Masashi, Oshimura Mitsuo, Ohbayashi Tetsuya

机构信息

Research Center for Bioscience and Technology Tottori University Yonago Japan; Japan Society for the Promotion of Science Tokyo Japan.

Chromosome Engineering Research Center Tottori University Yonago Japan; Division of Human Genome Science Department of Molecular and Cellular Biology School of Life Sciences Faculty of Medicine Tottori University Yonago Japan.

出版信息

FEBS Open Bio. 2017 Jan 28;7(3):306-317. doi: 10.1002/2211-5463.12169. eCollection 2017 Mar.

DOI:10.1002/2211-5463.12169
PMID:28286726
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5337897/
Abstract

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.

摘要

用于基因敲除或谱系追踪的目标DNA序列的位点特异性切除是研究生物系统的有力工具。目前，位点特异性重组酶（SSR），如Cre或Flp重组靶盒，已通过单一SSR成功切除或倒置以调节转基因表达。然而，使用单一SSR可能会限制基因表达的复杂控制。本研究调查了使用三种不同整合酶系统（TP901-1、R4和Bxb1）扩展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，其中荧光素酶表达可通过位点特异性重组转换为荧光蛋白。可由不同整合酶独立调控的单个盒串联连接，并插入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靶向荧光素酶活性丧失，荧光被激活。此外，整合酶系统能够特异性切除靶向DNA序列，而不会与其他重组靶点发生交叉反应。这些结果表明，在MAC载体的特定位点联合使用这些整合酶系统可实现转基因表达的多重调控，并可能有助于基因组或细胞工程。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/994ae3e79a97/FEB4-7-306-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/31affe3c83fb/FEB4-7-306-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/a8debc76e0c7/FEB4-7-306-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/f3890bbda4c9/FEB4-7-306-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/19db47ae95cb/FEB4-7-306-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/994ae3e79a97/FEB4-7-306-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/31affe3c83fb/FEB4-7-306-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/a8debc76e0c7/FEB4-7-306-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/f3890bbda4c9/FEB4-7-306-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/19db47ae95cb/FEB4-7-306-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/11fb/5337897/994ae3e79a97/FEB4-7-306-g005.jpg

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