An accumulative site‐specific gene integration system using cre recombinase‐mediated cassette exchange

Kameyama, Yujiro; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

doi:10.1002/bit.22619

Cited by 47 publications

(37 citation statements)

References 32 publications

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“…Cre, Flp, and the φC31 serine integrase have been used for this purpose, eliminating the need to identify incompatible crossover sites for a single recombinase that will resist cassette excision over extended time periods. Schemes for executing successive rounds of RMCEbased integration have also been reported, where each cassette exchange results in inactivation of one product site, but introduction of a new site for use in subsequent reactions (101,102).…”

Section: Turning Cre Into An Integrasementioning

confidence: 99%

Cre Recombinase

Duyne

2015

Microbiol Spectr

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The use of Cre recombinase to carry out conditional mutagenesis of transgenes and insert DNA cassettes into eukaryotic chromosomes is widespread. In addition to the numerous in vivo and in vitro applications that have been reported since Cre was first shown to function in yeast and mammalian cells nearly 30 years ago, the Cre-loxP system has also played an important role in understanding the mechanism of recombination by the tyrosine recombinase family of site-specific recombinases. The simplicity of this system, requiring only a single recombinase enzyme and short recombination sequences for robust activity in a variety of contexts, has been an important factor in both cases. This review discusses advances in the Cre recombinase field that have occurred over the past 12 years since the publication of Mobile DNA II. The focus is on those recent contributions that have provided new mechanistic insights into the reaction. Also discussed are modifications of Cre and/or the loxP sequence that have led to improvements in genome engineering applications.

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Section: Turning Cre Into An Integrasementioning

confidence: 99%

Cre Recombinase

Duyne

2015

Microbiol Spectr

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“…These results reasonably explained that sufficient Cre was necessary for RMCE in the target cells and that sufficient gag-pol protein was necessary for producing viral virions from viral-packaging cells. In this study, much less amount of Cre protein might be delivered to cells compared with using the Cre-expression vector in our previous report (Kameyama et al, 2010). Nonetheless, Cre-mediated transgene integration was possible using Cre-IDRV.…”

Section: Discussionmentioning

confidence: 66%

“…Hybrid viral vectors could target a transgene into GSH sites such as CCR5 (Holt et al, 2010) and AAVS1 loci. We have also developed an accumulative integration system of transgenes using Cre and mutated loxPs (Kameyama et al, 2010;Kawabe et al, 2012;Obayashi et al, 2012). Therefore, if a loxP site for transgene integration is introduced into a GSH site using hybrid viral vectors, Cre-IDRV could be retargeted for repeated or accumulative integration of transgenes into the GSH site.…”

Section: Discussionmentioning

confidence: 99%

Targeted transgene insertion into the CHO cell genome using Cre recombinase‐incorporating integrase‐defective retroviral vectors

Kawabe

Shimomura

Huang

et al. 2016

Biotech & Bioengineering

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Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre-based cell engineering. Biotechnol. Bioeng. 2016;113: 1600-1610. © 2016 Wiley Periodicals, Inc.

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“…Furthermore, Cre has been reported to catalyze recombination at loxP sites having a certain degree of homology with the wild-type loxP (Lee and Saito 1998;Sauer 1992;Sauer 1996;Thyagarajan et al 2000). Mutant lox sites have been developed to increase the efficiency of Cre-mediated insertion to avoid re-excision (Albert et al 1995;Araki et al 1997;Liu et al 2007;Chatterjee et al 2010;Kameyama et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Site-specific integration of transgene targeting an endogenous lox-like site in early mouse embryos

et al. 2010

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Functional lox-like sequences have been identified within the yeast and mammalian genome. These hetero-specific lox sites also allow Cre recombinase to specifically target efficient integration of exogenous DNA into the endogenous pseudo-lox (=lox) sequences that occur naturally in the host genome using a Cre/loxP integrative recombination system. We investigated whether the Cre/=lox system is useful for site-specific integration of transgenes and for improving the production efficiency of transgenic animals. This is the first report on Cre-mediated integrative recombination targeting an endogenous lox-like sequence termed pseudo-loxm5 (=loxm5) in early mouse embryos. We characterized the Cre/=loxm5 system in embryonic environment. Cre-expressing plasmid and a transgene (CMV/LacZ gene) flanked by =loxm5 and =loxcorem5 sites were co-microinjected into the pronucleus of fertilized mouse oocytes. The injected eggs were transferred into foster mothers, and the recombination products were investigated. The results show that the =loxm5 site is an active substrate for Cre-mediated recombination in the mouse embryonic environment. The transgenesis efficiency was up to 27% (6/22). The site-specific integration of the transgene into the endogenous =loxm5 site was found in 50 % of the transgenic pups. Our findings demonstrated that the Cre/=loxm5 integrative recombination system is an efficient and simple strategy for targeting an endogenous lox-like site in mammalian embryos.

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An accumulative site‐specific gene integration system using cre recombinase‐mediated cassette exchange

Cited by 47 publications

References 32 publications

Cre Recombinase

Cre Recombinase

Targeted transgene insertion into the CHO cell genome using Cre recombinase‐incorporating integrase‐defective retroviral vectors

Site-specific integration of transgene targeting an endogenous lox-like site in early mouse embryos

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