Site-specific Incorporation Of A Skeletal Muscle-specific Enhancer In The Regulatory Region Of Igf1 Upregulates IGF1 Expression

In the last decades,many genetically modified(GM)animals have been generated.However,the traditional method of creating transgenic animals may lead to random incorporation of the coding cassette into the genome.Although this technique has been shown to be simple and straightforward,the copy number and the insertion sites of the transgenes cannot be accurately controlled,thus highly likely leading to unstable expression of foreign genes and disturbing the expression of the endogenous genes.To circumvent the drawbacks of random insertion,site-specific incorporation of the expressing cassette via homologous recombination has been widely used as an improved option,and thus foreign genes can be specifically incorporated into a desired locus in a single copy.However,this approach is very arduous particularly when genomic DNA sequences of genes that span large genomic regions are used.Although cDNA can be used as a substitute of genomic DNA for the construction of targeting vectors,it may lack complex non-coding regulatory elements which may be undefined but of significant importance,and thus may be unfavourable for high expression levels in many cases.IGF1 is a key factor regulating skeletal muscle development and growth,and muscle specific up-regulation of IGF1 leads to muscle hypertrophy.Until recently,no Igfl transgenic mice have been generated using its full genomic DNA,which is as large as>70 Kb.To our knowledge,the existing Igfl transgenic mouse models have all been created via the random insertion of the cDNA construct encoding either different isoforms of pre-pro-peptide or the mature form.In this study,I used Igfl gene as an example in this proof-of-concept investigation.I asked whether the expression of Igfl can be upregulated by incorporating an enhancer into its non-coding regions upstream of its transcriptional start site;also we sought to investigate whether this genetic modification would lead to the expected phenotype in the mouse model.In this study,I used the luciferase reporter assays to evaluate the activities of the MLC(Myosin light chain)enhancer and MCK(Muscle creatine kinase)enhancer in enhancing the Igfl promoter activity.In the differentiated C2C12 myotubes,the MCK enhancer caused a slight increase in the luciferase activity(MCK enhancer in 3'-5' direction,P<0.05;MCK enhancer in 5'-3' direction,P>0.05),while the MLC enhancer dramatically increased the luciferase expression by 12 to 20-fold(MLC enhancer in 3'-5' direction,P<0.01;MLC enhancer in 5'-3' direction,P<0.05).So I chose the MLC enhancer for the subsequent research.To select an appropriate site for the incorporation of the MLC enhancer,I calculated the phastCons scores of the 100 Kb region upstream of Igfl transcriptional start site,and chose the three candidate sites with the lowest PhastCons average scores.In another words,these regions are the least evolutionarily conserved sites between different species,which are less likely to be functional cis-regulatory elements that are generally evolutionarily conserved.The three sites were named si tel,site2 and site3,which are approximately 1.5 Kb,6 Kb and 20 Kb far from the Igfl transcriptional start site,respectively.To evaluate the three candidate incorporation sites unbiasedly,I generated C2C12 single-cell colonies harbouring the MLC enhancer in each of the three sites by using the CRISPR/Gas9 technology and examined the expression levels of Igfl in myotubes of the three series of single-cell colonies.The expression of Igfl mRNA were down-regulated in single-cell colonies of both sitel and site3 series and only single-cell colonies of the site2 series displayed a dramatic upregulation of both Igfl mRNA and IGF1 protein.This result demonstrated that the site2 can support the incorporated MLC enhancer to exert its activity on Igfl promoter.I co-injected the sgRNA targeting site2,Cas9 mRNA and the homologous recombination vector into the mouse zygote and obtained the genetically modified mouse model.Both the male and female GM mice developed normally and exhibited dramatically elevated Igfl mRNA(P<0.0001)and IGF1 protein levels(P<0.000001)in the skeletal muscles.Interestingly,the IGF1 protein levels in the muscle of homozygous female mice was 12.4 fold compared with that in the wild type(WT)female littermates,while the IGF1 protein levels in the muscle of homozygous male mice was 4.6 fold compared with that in the WT male littermates.The GM female mice exhibited conspicuous muscle hypertrophy and the GM male mice also exhibited a similar trend of increased muscle weight and muscle hypertrophy,albeit not significant in statistics(P>0.05).Compared with their WT littermates,the GM mice showed no differences in their circulating IGF1 levels(P>0.05)and no heart abnormalities.There's no obvious increase in the tibia length in the GM mice(P>0.05).Our findings demonstrate that the site-specific incorporation of an enhancer in non-coding regions is a feasible method of upregulating gene expression levels and obtaining animals with desired traits.Our research provides a new strategy for creating GM animals and paves the way for the improvement of the production traits of livestock.