PhiC31-based Site-Specific Transgenesis System for Production of
Transgenic Bovine Embryos by Somatic Cell Nuclear Transfer
and Intracytoplasmic Sperm Injection

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Abstract

Objective

The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection.

Materials and Methods

In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer (SMGT) approaches.

Results

PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage (attP) sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction (PCR) and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique.

Conclusion

The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein ( EGFP) positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm injection (ICSI) method.

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Most cited references 39

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PiggyBac transposon-mediated gene transfer in human cells.

Matthew Wilson, Craig J Coates, Alfred George (2007)

Transposons are mobile genetic elements that can be used to integrate transgenes into host cell genomes. The piggyBac transposon system has been used for transgenesis of insects and for germline mutagenesis in mice. We compared transposition activity of piggyBac with Sleeping Beauty (SB), a widely used transposon system for preclinical gene therapy studies. An engineered piggyBac transposon with minimal length 5' and 3' terminal repeats exhibited greater transposition activity in transfected cultured human cells than a well-characterized hyperactive SB system. PiggyBac excision was very precise as evidenced by the typical absence of "footprint" mutations at the site of transposon excision. We mapped 575 piggyBac integration sites in human cells to determine site selectivity of genomic integration. PiggyBac demonstrated non-random integration site selectivity that differed from that previously reported for SB, including a higher preference for integrations in regions surrounding transcriptional start sites and within long terminal repeat elements. Importantly, overproduction inhibition was not observed with piggyBac, a major limitation of the SB system. This permitted the generation of combination "helper-independent" piggyBac transposase-transposon vectors that exhibited a 2-fold increase of transposition activity in human cells as compared with cells transfected with separate transposon and transposase plasmids. We conclude that piggyBac is a transposon system with certain properties, including high efficiency and lack of overproduction inhibition that are advantageous in preclinical development of transposon-based gene therapy.

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Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange.

Jack Bateman, Anne Lee, C.-ting Wu (2006)

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the C31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.

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A phage integrase directs efficient site-specific integration in human cells.

E-G Olivares, Michele Calos, Aaron A. Groth … (2000)

The integrase from the Streptomyces phage phiC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the phiC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.

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Author and article information

Journal

Journal ID (nlm-ta): Cell J

Journal ID (iso-abbrev): Cell J

Journal ID (publisher-id): Royan Institute

Title: Cell Journal (Yakhteh)

Publisher: Royan Institute

ISSN (Print): 2228-5806

ISSN (Electronic): 2228-5814

Publication date (Print): Spring 2018

Publication date (Electronic): 01 January 2018

Volume: 20

Issue: 1

Pages: 98-107

Affiliations

[1 ] Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran

[2 ]Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

[3 ]Department of Biology, Facualty of Sciences, Uneversity of Isfahan, Isfahan, Iran

[4 ]Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran

Author notes

[*Corresponding Address: ]P.O. BOX: 8165131378Department of Reproductive BiotechnologyReproductive Biomedicine Research CenterRoyan Institute for Biotechnology ACECRIsfahanIran Email: mh.nasr-esfahani@ 123456royaninstitute.org

Article

Other ID: Cell-J-20-98

DOI: 10.22074/cellj.2018.4385.

PMC ID: 5759686

PubMed ID: 29308625

SO-VID: b54f945b-9155-4df1-be17-c678e01d6e24

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

History

Date received : 19 June 2016

Date accepted : 1 March 2017

Custom metadata

Citation Sekhavati MH, Hosseini SM, Tahmoorespur M, Ghaedi K, Jafarpour F, Hajian M, Dormiani K, Nasr-Esfahani MH. PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection. Cell J. 2018; 20(1): 98-107. doi: 10.22074/cellj.2018.4385.

Keywords: intracytoplasmic sperm injection,somatic cell nuclear transfer,transgenesis

Data availability:

Keywords: intracytoplasmic sperm injection, somatic cell nuclear transfer, transgenesis

PhiC31-based Site-Specific Transgenesis System for Production of Transgenic Bovine Embryos by Somatic Cell Nuclear Transfer and Intracytoplasmic Sperm Injection

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Abstract

Objective

Materials and Methods

Results

Conclusion

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SDG: 12: Responsible Consumption and Production

Most cited references 39

PiggyBac transposon-mediated gene transfer in human cells.

Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange.

A phage integrase directs efficient site-specific integration in human cells.

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