Construction Of Specific Transgenic Vector Of Alp-MC1R For Melanocyte Of Sheep

MC1R (Melanocortin-1 Receptor) is proved by many studies to be the major gene to control the coat color of the mammals. This study connects the MC1R of alpaca to Lentiviral vector in order to build a transgenic expression vector.Then let the vector transfect the melanocytes of sheep.The vector will specifically express in the melanocytes of sheep.Then we will identify the activity and function of the vector from the level of mRNA and protein.It will lay the foundation for the transgenic sheep who will express the coat color of Alpaca.(1) According to the gene sequence of MC1R of alpaca (GenBank:FJ517582.2) which is loaded on NCBI, design specific primers with restriction enzyme cutting site.The primers are sure to amplify the whole CDS region of MC1R (1069bp).The sequencing result of the product of PCR has 99% registration with the MC1R gene in other species.The product of PCR can be connected to the vector in the next.(2) The MC1R gene is digested with Sal and Xba I.The Lentiviral vector is also digested with Sal and Xba I.Then connect the two gene fragments to together with T4 DNA Ligase.The sequencing result is right.(3)We use the recombinant plasmid to transfect the melanocytes of sheep.After 36h, we can observe the green fluorescent light with the fluorescence microscope.After 48h, we extract the RNA and the total protein of the melanocytes.(4)The expriment sets three groups:experimental group(transfecting recombinant plasmid), Negtive Control group(transfecting empty gsensor of Lentiviral vector), blank group(no transfection).Then QRT-PCR is used to analyze the mRNA expression level of gene MC1R, MITF, TYR and TYRP2 in the three groups.The result shows that the expression of MC1R in experimental group is 3.193 times of the expression in blank group.As for MITF, the value is 2.021.TYR is 3.218 and TYRP2 is 3.140. The exression level discrepancy of mRNA of MC1R, MITF, TYR, TYRP2 in experimental group and blank group are significant.(5) Western blot is used to analysis the average protein content of MITF, TYR and TYRP2 in experimental group, Negtive Control group and blank group.The result shows that the average protein content of the MITF in experimental group is 1.852 times of which is in the blank group.TYR is 3.537 and TYRP2 is 2.045.The average protein content differences between the in experimental group and the blank group are significant.In conclusion, after the transfection of the transgenic expression vector of MCI R of alpaca, the mRNA expression level of MC1R, MITF, TYR and TYRP2 which act as the major genes controling the activity of melanocytes and synthesis of melanin improves significantly.The average protein content of MITF, TYR and TYRP2 also improves significantly. Transgenic expression vector of MCIR of alpaca is successfully constructed and its effect is good.