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.
Highlights
Programmable nucleases like Clustered regularly interspaced short palindromic repeats/Cas9 (CRSIPR/Cas9), transcription activator-like effector nuclease (TALEn) and Zinc-finger nucleases (ZFN) have made considerable contributions to the development of precision gene editing and transgenics, but up to date they still face limitations due to a shortage of known sites for reliable and sustained transgene expression in transgenic livestock [1]
As shown in previous work [7], φC31 integrase is capable of catalyzing site-specific DNA recombination between an attB-containing donor plasmid and a pseudo attP site in mammalian genomes to form a single-copy and unidirectional genetic structure, which is believed to be beneficial to transgene expression [8,9,10]
A range of different amounts of φC31 integrase mRNA combined with pBCPB+ plasmid were microinjected into the cytoplasm of porcine parthenogenetic embryos and the recombined attR and attL DNA sequences were detected by embryo-based PCR 48 h post microinjection when cleaved into 2-cell stage
Summary
Programmable nucleases like Clustered regularly interspaced short palindromic repeats/Cas (CRSIPR/Cas9), transcription activator-like effector nuclease (TALEn) and Zinc-finger nucleases (ZFN) have made considerable contributions to the development of precision gene editing and transgenics, but up to date they still face limitations due to a shortage of known sites for reliable and sustained transgene expression in transgenic livestock [1]. Up to now there has been no powerful approach available to identify novel safe sites in vivo at genome-wide level To address this issue, we took advantage of φC31 integrase to integrate a reporter gene into pseudo attP sites of the pig genome so as to assess the expression potential of these sites. We isolated two novel pseudo attP sites in the pig genome using φC31 integrase and characterized them as safe harbors for site-specific transgene integration and robust transgene expression. These sites add to our repertoire and provide new options to stably integrate a wide range of transgenes for robust and predictable expression. We envision that these novel safe harbors will facilitate the creation of transgenic pigs for agricultural and biomedical applications
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