| Objective: (1)To construct the transgenic expression vector of human matrix metalloproteinases-12(hMMP-12) with chromatin insulator which can be specifically expressed in macrophages; (2)Creation and characterization of hMMP-12 transgenic rabbits. Methods: (1)The full length hMMP-12 DNA was obtained by using RT-PCR and was subcloned into pGEM-T vector and the subclone was named as pGEM-T-hMMP-12 after DNA sequencing;(2) Subcloning pJC13-SR which has a promoter and a enhancer of human scavenger receptor A,a human growth hormone tail and chromatin insulators;(3)Macrophage-specific expression vector was cloned by using pGEM-T-hMMP-12 and pJC13-SR;(4)After linearization of pGEM-T-hMMP-12,it was used for producing transgenic rabbits by microinjection. PCR and Southern blotting analysis were applied for identification of transgenic rabbits. Results: (1)The hMMP-12 was successfully cloned into the needed vector by enzymatic digestion and DNA sequencing;(2)The total efficiency of the gene transfer was between 1.41% and 3.35% with integration rates of 20% and 50% by PCR analysis. The total efficiency of the gene transfer was between 0.47% and 2.23% with integration rates of 13.33% and 16.67% determined by Southern blotting. Conclusion: This work has laid foundation for the construction of hMMP-12 transgenic rabbit models and for further studied on the role... |
