Durable Expression of Minicircle DNA-Liposome-Delivered Androgen Receptor cDNA in Mice with Hepatocellular Carcinoma
Figure 1
Construction of AR-expressing minicircle vectors and the production of minicircle DNA. (a) Construction of AR-expressing minicircle DNA. Parental plasmid contained antibiotics kanamycin resistance gene and replication EcoE1 Ori cassette flanked by attP and attB site recognized by φC31 intergrase. Upon addition of arabinose, φC31 intergrase and ISce1 endonuclease gene are activated by pBAD operon. The plasmid carries a CMV promoter driven GFP gene and multiple cloning sites (MCS) where AR cDNA is inserted. The 3.2 kbp open-reading frame human AR cDNA was cloned into BamH1 site on MCS to produce a 10 kb AR-expressing plasmid. While producing minicircle DNA, φC31 facilitates the recombination of attP and attB sites and produces two smaller circular DNA fragments, minicircle and bacterial backbone. The backbone plasmid was linearized by ISce1 and further degraded by bacterial endogenous DNase, resulting in a transgene vector (minicircle) without plasmid ori and antibiotic resistance genes. (b) GFP minicircle parental plasmids with backbone (pMCP.GFP, 7 kbps; left panel, 2nd lane) and the minicircle DNA (MC.GFP, 3 kbps; right panel, 2nd lane). AR minicircle parental plasmids with backbone (pMCP.hAR, 10 kbps; left panel, 3rd lane) and the minicircle DNA (MC.hAR, 6 kbps; right panel, 1st lane). Reference molecular weight marker for parental plasmids is shown on the left-hand side of the panel. Reference molecular weight marker for minicircle DNAs is shown on the right-hand side of the panel.
