Dataset Information

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome.

ABSTRACT:

Background

Phage-encoded serine integrases, such as ?C31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays.

Results

We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; ?C31 integrase.

Conclusions

We conclude that the Bxb1 and ?C31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase.

SUBMITTER: Xu Z

PROVIDER: S-EPMC4015280 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Publications

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome.

Xu Zhengyao Z Thomas Louise L Davies Ben B Chalmers Ronald R Smith Maggie M Brown William W

BMC biotechnology 20131020

<h4>Background</h4>Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays.<h4>Results</h4>We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases we ...[more]

PMID: 24139482

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Project description:Recombinant Chinese hamster ovary (CHO) cell line development for complex biotherapeutic production is conventionally based on the random integration (RI) approach. Due to the lack of control over the integration site and copy number, RI-generated cell pools are always coupled with rigorous screening to find clones that satisfy requirements for production titers, quality, and stability. Targeted integration into a well-defined genomic site has been suggested as a possible strategy to mitigate the drawbacks associated with RI. In this work, we employed the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system in combination with the Bxb1 recombinase-mediated cassette exchange (RMCE) system to generate an isogenic transgene-expressing cell line. We successfully utilized the CRIS-PITCh system to target a 2.6 kb Bxb1 landing pad with homology arms as short as 30 bp into the upstream region of the S100A gene cluster, achieving a targeting efficiency of 10.4%. The platform cell line (PCL) with a single copy of the landing pad was then employed for the Bxb1-mediated landing pad exchange with an EGFP encoding cassette to prove its functionality. Finally, to accomplish the main goal of our cell line development method, the PCL was applied for the expression of a secretory glycoprotein, human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2). Taken together, on-target, single-copy, and stable expression of the transgene over long-term cultivation demonstrated our CRIS-PITCh/RMCE hybrid approach might possibly improve the cell line development process in terms of timeline, specificity, and stability. KEY POINTS: • CRIS-PITCh system is an efficient method for single copy targeted integration of the landing pad and generation of platform cell line • Upstream region of the S100A gene cluster of CHO-K1 is retargetable by recombinase-mediated cassette exchange (RMCE) approach and provides a stable expression of the transgene • CRIS-PITCh/Bxb1 RMCE hybrid system has the potential to overcome some limitations of the random integration approach and accelerate the cell line development timeline.

Dataset Information

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome.

Background

Results

Conclusions

Publications

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome.

Similar Datasets

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