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Minicircle Vectors for Transgene and miRNA Expression

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The development of safe and efficient vector systems is a crucial step for successful gene transfer in eukaryotic cells.

Minicircle vectors are novel super-coiled minimal expression cassettes that are derived from conventional plasmid DNA by site-specific recombination in vivo in E. coli.  Minicircle vectors do not carry antibiotic resistance genes, nor bacterial origin of replication, thus markedly reducing immune and inflammatory response and effectively protecting transgene expression from being silenced due to the bacterial backbone sequences. Minicircle vectors are generated from parental plasmids in vivo in E. coli through site-specific recombination.  The parental plasmids carry the eukaryotic expression cassettes flanked by two mutant loxP sites (i.e., mutation of the terminal 5 nucleotides on each side) of site-specific Cre recombinase and are transformed into the engineered E. coli strain expressing the Cre recombinase under control of the pBAD/araC promoter.

L-arabinose effectively induces expression of Cre recombinase in  the bacterial strain, resulting in the excision of the interjacent DNA sequences. The recombination between the two loxP sites promotes the division of the parental plasmid into two supercoiled DNAs:  (1) a minicircle vector that carries eukaryotic expression cassette for transgenes and miRNAs and a double mutant loxP site, and (2) a replicative miniplasmid containing the bacterial backbone sequences and a wild-type loxP site. The formation of double mutant loxP site compromises the recognition of the sequences by the Cre recombinase, resulting in a shift in the equilibrium towards minicircle vector production. Following the induction of Cre recombinase, the different forms of supercoiled DNA are isolated from the bacterial lysates and subjected to restriction enzyme digestion to linearize parental plasmid and miniplasmid. Subsequently, the minicircle vectors are further purified using a cesium chloride gradient.