Dataset Information

The ?BT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia.

ABSTRACT: Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages ?C31 and ?BT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the ?BT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains; however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on ?BT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a ?BT1 integrase-based vector can insert at multiple pseudo-attB sites within a single strain and we determine the sequence of a pseudo-attB motif. These data suggest that ?BT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.

SUBMITTER: Herisse M

PROVIDER: S-EPMC5937489 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia.

Herisse Marion M Porter Jessica L JL Guerillot Romain R Tomita Takehiro T Goncalves Da Silva Anders A Seemann Torsten T Howden Benjamin P BP Stinear Timothy P TP Pidot Sacha J SJ

PeerJ 20180504

Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages ΦC31 and ΦBT1. ...[more]

PMID: 29740520

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Project description:Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5' and 3' junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.

Dataset Information

The ?BT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia.

Publications

The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-<i>attB</i> sites in the genus <i>Nocardia</i>.

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