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Int. J. Mol. Sci. 2018, 19(1), 149; https://doi.org/10.3390/ijms19010149

ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome

1
Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Institute of Animal Science and Veterinary Medicine, Hubei Academy of AgroSciences, Wuhan 430064, China
2
AgResearch Ltd., Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand
3
AgResearch Ltd., Ruakura Research Centre, Private Bag 3123, Hamilton 3214, New Zealand
*
Authors to whom correspondence should be addressed.
Received: 19 November 2017 / Revised: 25 December 2017 / Accepted: 1 January 2018 / Published: 4 January 2018
(This article belongs to the Section Biochemistry)
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Abstract

Programmable nucleases have allowed the rapid development of gene editing and transgenics, but the technology still suffers from the lack of predefined genetic loci for reliable transgene expression and maintenance. To address this issue, we used ФC31 integrase to navigate the porcine genome and identify the pseudo attP sites suitable as safe harbors for sustained transgene expression. The combined ФC31 integrase mRNA and an enhanced green fluorescence protein (EGFP) reporter donor were microinjected into one-cell zygotes for transgene integration. Among the resulting seven EGFP-positive piglets, two had transgene integrations at pseudo attP sites, located in an intergenic region of chromosome 1 (chr1-attP) and the 6th intron of the TRABD2A gene on chromosome 3 (chr3-attP), respectively. The integration structure was determined by TAIL-PCR and Southern blotting. Primary fibroblast cells were isolated from the two piglets and examined using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA), which demonstrated that the chr1-attP site was more potent than chr3-attP site in supporting the EGFP expression. Both piglets had green feet under the emission of UV light, and pelleted primary fibroblast cells were green-colored under natural light, corroborating that the two pseudo attP sites are beneficial to transgene expression. The discovery of these two novel safe harbors for robust and durable transgene expression will greatly facilitate the use of transgenic pigs for basic, biomedical and agricultural studies and applications. View Full-Text
Keywords: ФC31 integrase; pseudo attP site; safe harbor; transgene; pig ФC31 integrase; pseudo attP site; safe harbor; transgene; pig
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Bi, Y.; Hua, Z.; Ren, H.; Zhang, L.; Xiao, H.; Liu, X.; Hua, W.; Mei, S.; Molenaar, A.; Laible, G.; Zheng, X. ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome. Int. J. Mol. Sci. 2018, 19, 149.

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