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.
Highlights
New genes are routinely introduced to mammalian cells to elucidate their expression pattern and function
After verification of whether the candidate loci were suitable for sustainable foreign gene expression on cellular and individual level, we considered a candidate site Pifs[501] as a potential effective transgene integration site which could be used in pig genome engineering
Efficient transgene expression in recipient cells constitutes the primary step in animal model construction and ensures the emergence of relevant phenotypes
Summary
New genes are routinely introduced to mammalian cells to elucidate their expression pattern and function This is often achieved by random integration into the genome, either by viral transduction[1,2] or plasmid transfection. Uncontrolled transgene integration can lead to several undesirable effects, including unpredictable expression and unexpected interactions between integrated exogenous DNA and the neighboring chromatin environment[4,5] It would be problematic once transgenes activate oncogene expression[6]. Intergenic regions within epigenetic open chromosome regions might support high and reliable expression of transgene This would be similar to the identification of intergenic H11 locus, where neighboring genes displayed broad spatial and temporal EST expression patterns[27,28]
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