Abstract
In this study, we upregulated insulin-like growth factor-1 (IGF1) expression specifically in skeletal muscle by engineering an enhancer into its non-coding regions and verified the expected phenotype in a mouse model. To select an appropriate site for introducing a skeletal muscle-specific myosin light chain (MLC) enhancer, three candidate sites that exhibited the least evolutionary conservation were chosen and validated in C2C12 single-cell colonies harbouring the MLC enhancer at each site. IGF1 was dramatically upregulated in only the site 2 single-cell colony series, and it exhibited functional activity leading to the formation of extra myotubes. Therefore, we chose site 2 to generate a genetically modified (GM) mouse model with the MLC enhancer incorporated by CRISPR/Cas9 technology. The GM mice exhibited dramatically elevated IGF1 levels, which stimulated downstream pathways in skeletal muscle. Female GM mice exhibited more conspicuous muscle hypertrophy than male GM mice. The GM mice possessed similar circulating IGF1 levels and tibia length as their WT littermates; they also did not exhibit heart abnormalities. Our findings demonstrate that genetically modifying a non-coding region is a feasible method to upregulate gene expression and obtain animals with desirable traits.
Highlights
Many genetically modified (GM) animals with desirable phenotypes have been generated for agricultural and biomedical applications
The MLC27–30 and MCK31–34 enhancers are extensively characterized muscle-specific enhancers; we chose them as candidates for insertion into the upstream regions of the Igf[1] transcriptional start site
The site 2 series of single-cell colonies more readily differentiated into myotubes (Supplementary Fig. S5), and more myotubes were detected by immunofluorescence staining of myosin heavy chain (MHC) (Fig. 4). These results showed that site 2 was an ideal site for incorporation of the myosin light chain (MLC) enhancer to drive Igf[1] expression in C2C12 cells
Summary
Many genetically modified (GM) animals with desirable phenotypes have been generated for agricultural and biomedical applications. Most traditional methods for creating transgenic animals usually involve the random insertion of the coding cassette into the genome[1,2,3,4,5]. This technique seems straightforward, the copy number and insertion locus cannot be accurately controlled, leading to unstable gene expression[6,7]. Most studies focus on naturally occurring mutations, and reports of artificially editing non-coding genomic sequences to improve production traits in live animals are rare. We investigated whether this genetic modification generates the expected phenotype in the mouse model
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