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

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.

High fidelity one‐pot DNA assembly using orthogonal serine integrases

“…Tomimatsu et al . [ 52 ] used Bxb1, ϕR4, and TP901 integrases to mediate multiple cassette exchange on a mouse chromosome. Some in vivo applications have used central dinucleotides as the basis for specific DNA assembly.…”

Engineering Synthetic Chromosomes by Sequential Loading of Multiple Genomic Payloads over 100 Kilobase Pairs in Size

“…The ACE engineering system provides a tractable engineering platform for transferring large (>100 kbp) genetic payloads with native genetic architecture onto a synthetic chromosome. Although other MAC platforms have been developed for multiple loadings onto a synthetic mammalian chromosome,2, 4, 6, 27, 28 these previously described platforms required the creation of multiple vectors with different site-specific recombination systems to achieve multiple loadings and have not reached the engineerable carrying capacity demonstrated here. For example, Honma et al 28 .…”

Engineered Bacteriophages for Practical Applications

“…This organism can utilize carbon monoxide and carbon dioxide, but until now there were few genetic manipulation tools available. 71) In a similar fashion, these recombinases are being used to study different eukaryotes. For example, a mouse artificial chromosome was modified so it could be edited by three different phage integrases.…”

“…The transient expression of one of said integrases allowed the specific editing of its target sequence without affecting the targets of the other integrases. 72) One of these integrases was also shown to work in Drosophila melanogaster, adding to the classical tools available for this valuable model species. 73) These results show that phages as a source of new tools for genetic engineering and molecular biology are far from being exhausted, with likely much more to come from yet unknown phages.…”