An In Vivo Robust System For Generation Of Site-Specific Integration Minicircle DNA Vector

Transgenic technology in the field of gene therapy,transgenic animals,genome modification and other biomedical research has an important application value.There are a variety of techniques available for transgenesis,the classic transgenic approaches for producing transgenic animals usually lead to random integration of exogenous genes,single point multiple copy integration,lower levels of transgene expression and bacterial backbone sequence redundancy integration.In order to solve the above problems,this study established a time-saving and efficient method for obtaining site-specific integrated mimicircle DNA by combining the LR clonase system and the Streptomyces phage ?C31 integrase system.A parental plasmid containing LR clonase system and ?C31 integrase system is constructed,it can be recombined by L-arabinose induced LR clonase in bacteria to produce a miniplasmid expressing ?C31 integrase in eukaryotic cells and a minicircle DNA containing the original genes such as the target gene and attB site.The LR clonase system could effectively catalyze the recombination of ? attL and?attR in the parental plasmid in bacteria,and the recombination rate was more than91%,compared to commercialized LR clonase in vitro reaction,it has a higher recombination rate with a more efficient and more economical manner.After purification,the mix of miniplasmid and minicircle DNA could be successfully cotransfected into HeLa cells without further isolation,eliminating the loss of minicircle DNA caused by the seperation,while reducing the experimental steps and saving time,reducing the cost of the preparation.The ?C31 integrase in the miniplasmid can effectively mediate the integration of the minicircle DNA carrying exogenous gene and the attB sequence into the mammalian genome "pseudo attP" site in mammalian cells to achieve the site-specific and single copy integration of the target gene.Furthermore,the parental plasmid we constructed in this study can produce minicircle DNA with excisable screening gene and markers to reduce labor for postive colonies collection which will expand the application scope of minicircle DNA in transgenetic animals.