Dataset Information

Screen and Verification for Transgene Integration Sites in Pigs.

ABSTRACT: Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites in pig genome. Using an in silico approach, we screen out two candidate sites, namely, Pifs302 and Pifs501, located in actively transcribed intergenic regions with low nucleosome formation potential and without potential non-coding RNAs. After CRISPR/Cas9-mediated site-specific integration on Pifs501, we detected high EGFP expression in different pig cell types and ubiquitous EGFP expression in diverse tissues of transgenic pigs without adversely affecting 600?kb neighboring gene expression. Promoters integrated on Pifs501 exhibit hypomethylated modification, which suggest a permissive epigenetic status of this locus. We establish a versatile master cell line on Pifs501, which allows us to achieve site-specific exchange of EGFP to Follistatin with Cre/loxP system conveniently. Through in vitro and in vivo functional assays, we demonstrate the effectiveness of this screening method, and take Pifs501 as a potential site for transgene insertion in pigs. We anticipate that Pifs501 will have useful applications in pig genome engineering, though the identification of genomic safe harbor should over long-term various functional studies.

SUBMITTER: Ma L

PROVIDER: S-EPMC5943519 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Screen and Verification for Transgene Integration Sites in Pigs.

Ma Linyuan L Wang Yuzhe Y Wang Haitao H Hu Yiqing Y Chen Jingyao J Tan Tan T Hu Man M Liu Xiaojuan X Zhang Ran R Xing Yiming Y Zhao Yiqiang Y Hu Xiaoxiang X Li Ning N

Scientific reports 20180509 1

Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites in pig genome. Using an in silico approach, we screen out two candidate sites, namely, Pifs302 and Pifs501, located in actively transcribed intergenic regions with low nucleosome formation potential and witho ...[more]

PMID: 29743638

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Project description:BackgroundThe ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei. Currently, only the locus of exocellobiohydrolases I encoding gene (cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized.ResultsTo search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified.ConclusionsWe have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei. Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.

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

Screen and Verification for Transgene Integration Sites in Pigs.

Publications

Screen and Verification for Transgene Integration Sites in Pigs.

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