A Mouse Model Construction Of IGF-1 C.258A>G Synonymous Mutation And Its Function Analysis

In addition to the restriction of environmental,nutritional and other factors,the growth and development regulation of animals is also subject to long-term genetic selection.Among them,the IGF-1 gene in the neuroendocrine growth axis is considered to play a vital role in the body growth.The unique physiological characteristics of miniature pigs have received wide attention from the biomedical community,but the formation mechanism of the dwarf size of miniature pigs has not been elucidated yet.In order to explore the potential regulatory mechanism of body-size formation,Cheng et al.screened out only one synonymous mutation c.258A>G in the exon 4 of IGF-1gene,on the basis of observing differences of IGF-1 expression in pigs with different body types.Studies have shown that synonymous mutations can affect gene expression by various aspects of protein biosynthesis and also affect the biological function by changing the folding pattern of newborn peptide chains without changing amino acid types.However,the current understanding of the molecular mechanism of synonymous mutations is still limited to in vitro experiments.Therefore,in this study,the construction of single-base mutation mice model in vivo is of great significance for analyzing the mechanism of heritable variation in IGF-1 gene.With the continuous breakthrough of single-base editors,ABEmax system was used to construct the IGF-1 c.258A>G synonymous mutant mice here.After identifying the genotypes and off-target effects,the mice were stably inherited to F2 generation.In order to investigate whether the synonymous mutation affects IGF-1 gene expression,RT-q PCR and Western Blot were used to detect the IGF-1 expression in the liver of wild-type,heterozygous mutant and homozygous mutant mice at different growth and development stages.Then,ELISA was used to detect the secretion of IGF-1 in serum.Compared with wild-type mice,the expression level of IGF-1 in liver of homozygous mutant mice aged 6 weeks and 8 weeks showed a decreasing trend,but there was no significant difference(P>0.05).While the secretion level of serum IGF-1 in homozygous mutant mice was significantly decreased(P<0.05).At the same time,the growth curve and body length of the F2 generation wild-type,heterozygous mutant and homozygous mutant mice were monitored,and the viscera index of the 8-week-old mice was calculated.Additionally,the bone development was observed using X-ray and histological staining.The experimental results showed that there was no significant difference in the growth and development of whole-body among the mice of three genotypes,and there was no abnormal or deformed phenotype in the femoral epiphysis and growth plate.It was speculated that the synonymous mutation did not significantly down-regulate the IGF-1 content,may be compensated by other compensatory effects,which was not enough to cause large fluctuations in the body traits formation.To explore whether t RNA abundance and sequence context affect the function of synonymous codon,four synonymous codons were substituted for all Ala(the amino acid encoded by the codon of the synonymous mutation is alanine)in the IGF-1 mature peptide sequence.Additionally,an extra IGF-1 interchanged the position of GCG and GCA was synthesized and the IGF-1 mature peptide sequence in large pig breeds was taken as the wild type as the control.These six groups of IGF-1 recombinant vectors carrying different Ala synonymous codons were constructed by adding FLAG tags at the C-terminal.Different expression vectors were transfected into PK-15 cells and RTq PCR and Western Blot were used to detect the expression of FLAG.The results showed that the expression level of IGF-1 encoded by the four synonymous codons was consistent with the trend of codon preference and t RNA abundance,while was significantly lower than that of the IGF-1-WT group(P<0.05).In order to further explore the molecular mechanism of different Ala synonymous codons on IGF-1protein synthesis,this study tested the mRNA secondary structure and stability.The results showed that IGF-1 encoded by different synonymous codon combinations had different mRNA secondary structures,which affected the stability of IGF-1transcription and translation levels.The IGF-1 mRNA half-life period in the IGF-1-GCT group was basically similar to that in the IGF-1-WT group,however,the mRNA stability in the other groups was higher than that in the IGF-1-WT group to some extent.In addition,the detection of protein translation initiation rate and protein half-life indicated that IGF-1 carrying diverse synonymous codon combinations had different protein initiation efficiency.The IGF-1 protein half-life of IGF-1-WT group was the shortest,while the stability of IGF-1-GCG and IGF-1-GCC group was significantly higher than that of IGF-1-WT group(P<0.05).Furthermore,to verify whether the IGF-1 encoded by each expression vector interferes with the biological function of the gene,CCK-8 experimental results suggested that the substitution of Ala synonymous mutations in the IGF-1 coding region can significantly affect the proliferation of PK-15 cells.In summary,the above results evaluated the impact of the synonymous mutation IGF-1 c.258A>G on the gene expression and secretion of at the animal level,and further explored the molecular mechanisms of synonymous codons affecting gene expression and biological functions.And it was demonstrated that although the synonymous codons usage of Ala is different,the optimal combination can maintain a balance of relative advantages in gene expression,stability and biological function.