A robust system for production of minicircle DNA vectors

Abstract

Minicircle DNA vectors allow sustained transgene expression in quiescent cells and tissues. To improve minicircle production, we genetically modified Escherichia coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, ΦC31 integrase and I-SceI homing endonuclease. This bacterial strain produces purified minicircles in a time frame and quantity similar to those of routine plasmid DNA preparation, making it feasible to use minicircles in place of plasmids in mammalian transgene expression studies.

Figure 1. Comparison of the new and old minicircle systems

(a) An earlier version of the minicircle production system. (aI) Structure of the previous minicircle producer plasmid. BAD and araC, the promoter and the repressor gene of the inducible L–arabinose-araC.BAD system; øC31, bacteriophage øC31 integrase gene; attB and attP, the bacterial and phage attachment sites of the øC31 integrase; I-SceI, I-SceI homing endonuclease gene; I-SceIs, the I-SceI recognition site; AmpR, ampicillin resistance gene; ColE1, DNA replication origin. (aII) E. coli strain Top10 Invitrogen (Carlsbad, California) original strain used to produce minicircle. (aIII) Flowchart showing the minicircle production protocol. Each box represents a major step and the darkened boxes represent the steps required in addition to a routine plasmid production protocol. (b) The new minicircle system. (bI) Diagram of new minicircle parental plasmids and its conversion to minicircle DNA. pMC.hFIX, minicrcle producer plasmid; hFIX, human factor IX; sApoE, promoter/enhancer as described previously; KanR, kanamycin resistance gene. Upon L-arabinose induction, øc31 is expressed to mediate the formation of minicircle and plasmid backbone circle and I-SceI to induce the destruction of plasmid backbone circle. (bII) The genetic modifications of the minicircle producing bacterial strain ZYCY10P3S2T. 10P3S2T = (1) 10 copies of BAD.øC31 cassette, which were integrated in 3 loci of the bacterial genome: 2 tandem copies at the ΔendA locus (Supplementary Figure 4c), and 4 copies at the araD (Supplementary Figure 5a) and galK (Supplementary Figure 6a) each; (2) 3 tandem copies of BAD.I-SceI cassette, which were integrated at UMU locus (Supplementary Figure 3a) and (3) 2 constitutively expressing L-arabinose transporter genes: one was araE gene driven by an artificial promoter cp8, which presented in strain BW27783; the other was the bla-lacY A177C cassette, which was integrated at the lacY locus (Supplementary Figure 2); bla, beta-galactosidase gene promoter; lacY A177C, the missense mutant of lacY gene. (bIII) Flowchart showing the new minicircle production protocol. (c) Stepwise genetic modification of the bacterial genome to make the current ZYCY10P3S2T strain.

Figure 2. Improvement in minicircle quality and quantity by the new manufacturing system

(a) Strain BW27783 produced minicircle (MC) with enhanced purity. Minicircles were produced according to the protocol described previously. p2øC3.hFIX, minicircle producer plasmid (PP); BglII+EcoN1, two restriction enzymes used to restrict MC before electrophoresis; L-arab(%), percent of L-arabinose in the minicircle induction reaction; PB, plasmid backbone circle. (b) Strategy for inactivation of endA. pKanR.endA, the plasmid used to generate the Pme1-restricted targeting DNA fragment; KanR, kanamycin-resistance gene; attB and attP, the bacterial and phage attachment sites of bacteriophage øC31 integrase; boxed endA, PCR-generated 329- and 754-bp end A fragments; pBAD.Red, a plasmid expressing the bacteriophage Lambda homology recombination complex (Red) under the control of araC.BAD (BAD); p2øC31, a complementing plasmid encoding two copies of BAD.øC31 gene, one copy of BAD.I-SceI gene and one I-SceI site; BWΔendA, a strain derived from BW27783 with the endA interrupted. (c) Minicircle DNA integrity before and after disruption of the endA gene. Before disrupting endA, we observed repeatedly large variations in the degree of plasmid degradation as demonstrated in Figures 2a&c. Because the Endonuclease A is a membrane-bound enzyme, it was possible that its membrane release and activation varied during plasmid preparation. 32°C and 37°C, the incubation temperature; hr., hours; all reactions contained 1% L-arabinose. (d) Quality of the minicircle determined by gel analyses. Minicircle was made according to the simplified protocol outlined in Figure 1b and Supplementary Figure 9, DNAs were restricted before electrophoresis; (e) Yield of minicircle producer plasmids and minicircle vector DNAs. The yield was derived from triplicate 400-ml overnight cultures; PP, Minicircle producer plasmid; MC, minicircle; Wilcoxon rank sum test comparing the yield of minicircle and its minicircle producer plasmid: (I), p<0.05; (II), p>0.05. We used the following formula to convert the yield from mg/L to mol/L: mol/L=[yield (mg/L)×10E-3 gram/L]/[size (kb)×1000 ×330×2 gram/mol], where 330 is the average molecular weight of dNTP. The minicircle producer plasmid, pMC.RSV-hAAT is schematically illustrated in Supplementary Figure 4d, while the pMC.CMV.LGNSO was described in details in the Constructs section of the Online Methods.