Dataset Information

Global mapping of binding sites for phic31 integrase in transgenic maden-darby bovine kidney cells using ChIP-seq.

ABSTRACT: Background:?C31 integrase, a site-specific recombinase, can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. The sequence features of endogenous binding sites will help us to fully understand the site-specific recognition function by ?C31 integrase. The present study was aimed to uncover the global map of ?C31 integrase binding sites in bovine cells and analysis the features of these binding sites by comprehensive bioinformatics methods. Results:In this study, we constructed a ChIP-seq method that can be used to uncover the global binding sites by phiC31 integrase. 6740 potential ?C31 integrase binding sites were identified. A sequence motif was found that contains inverted repeats and has similarities to wild-type attP site. Using REPEATMASKER, we identified a total of 20,183 repeat-regions distributed in 50 repeat types for the 6740 binding sites. These sites enriched in "regulation of GTPase activity" of in the GO category of biological process and KEGG pathway of signal transmembrane transporter activity. Conclusion:This study is the first time to uncover the global map of binding sites for ?C31 integrase using ChIP-sequencing method and analysis the features of these binding sites. This method will help us to fully understand the mechanism of the site-specific integration function by phiC31 integrase and will potentially boost its genetic manipulations in both gene therapy and generation of transgenic animals.

SUBMITTER: Qu L

PROVIDER: S-EPMC6332687 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Global mapping of binding sites for phic31 integrase in transgenic maden-darby bovine kidney cells using ChIP-seq.

Qu Lijuan L Wang Lei L Zhu Xueyuan X Zhang Yan Y Ou Qiang Q Ma Aying A Sheng Fengying F Wei Xiaoqing X Dai Yue Y Li Guoting G Xie Shuwu S

Hereditas 20190114

<h4>Background</h4>ΦC31 integrase, a site-specific recombinase, can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. The sequence features of endogenous binding sites will help us to fully understand the site-specific recognition function by ΦC31 integrase. The present study was aimed to uncover the global map of ΦC31 integrase binding sites in bovine cells and analysis the features of these binding sites by comprehensive bioinformatics methods ...[more]

PMID: 30675136

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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

Global mapping of binding sites for phic31 integrase in transgenic maden-darby bovine kidney cells using ChIP-seq.

Publications

Global mapping of binding sites for phic31 integrase in transgenic maden-darby bovine kidney cells using ChIP-seq.

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