Minicircles are episomal DNA vectors that are produced as circular expression cassettes devoid of any bacterial plasmid DNA backbone. Their smaller molecular size enables more efficient transfections and offers sustained expression over a period of weeks as compared to standard plasmid vectors that only work for a few days.
The Minicircle DNA elements are generated by an intramolecular (cis-) recombination from a parental plasmid (PP) mediated by PhiC31 integrase. The full-size MC-DNA construct is grown in a special host E. coli bacterial strain ZYCY10P3S2T (catalog#MN900A-1). This highly engineered strain harbors an Arabinose-inducible system to express the PhiC31 integrase and the I-SceI endonuclease simultaneously. The ZYCY10P3S2T strain also contains a robust arabinose transporter LacY A177C gene. Adding arabinose to the media turns on the integrase and endonuclease genes. The PhiC31 integrase produces the MC-DNA molecules as well as PP-DNA from the full-size MC-DNA construct. The Sce-I endonuclease then degrades the PP-DNA backbone.
Robust system for mcDNA
- Multiple vector formats
- Works in vitro and in vivo
- Optimized E. coli production strain
The Parental Plasmid DNA (PP) contains several engineered I-SceI restriction sites that ultimately lead to the destruction of the PP-DNA but not the MC-DNA. The difference between MC and standard plasmid vectors is that the MC no longer contains the bacterial origin of replication or the antibiotic resistance markers. Sequences within the bacterial plasmid backbone contain signals for methylation and transgene silencing. Thus delivering only the minicircles to cells lengthens the expression of the transgene over traditional transient transfections of plasmids.
For dividing cells, expression of the minicircles lasts up to 14 days. For non-dividing cells, expression of the minicircles drops slightly after the first week, but then can continue expressing the transgenes for months.
Minicircle DNA System Products
|pMC.BESPX-MCS1 (empty vector)||10 ug||MN100A-1|
|pMC.BESPX-MCS2 (empty vector)||10 ug||MN100B-1|
|pMC.CMV-GFP-SV40PolyA (positive control)||10 ug||MN601A-1|
|Pre-made Minicircle CMV-GFP-SV40PolyA (positive control)||40 ug||MN601MC-1|
|pMC.CMV-rRFP-SV40PolyA (positive control)||10 ug||MN602A-1|
|Pre-made Minicircle CMV-rRFP-SV40PolyA (positive control)||40 ug||MN602MC-1|
|Mouse IL-23 Pre-made Minicircle DNA||100 ug||MN651MC-1|
|Mouse IL-23 Enhanced Episomal Expression Vector||100 ug||EEV651A-1|
|pMC.CMV-GFP-T2A-Puro-H1-Scramble (shRNA with GFP and Puro scramble control)||10 ug||MNSI000A-1|
|pMC.CMV-GFP-H1-BamHI-EcoRI (shRNA with GFP cloning vector)||10 ug||MNSI500A-1|
|pMC.CMV-GFP-Puro-H1-BamHI-EcoRI (shRNA with GFP and Puro cloning vector)||10 ug||MNSI505A-1|
|pMC.EF1-GFP-T2A-Puro-H1-BamHI-EcoRI (shRNA with GFP and Puro cloning vector)||10 ug||MNSI506A-1|
|Kanamycin solution (50mg/ml)||20 ml||MN800A-1|
|Arabinose induction solution (20%)||20 ml||MN850A-1|
|ZYCY10P3S2T E. coli Minicircle producer strain (competent, ready to transform)||5 vials||MN900A-1|
|MC-Easy Minicircle DNA Production Kit (does not include ZYCY10P3S2T producer E. coli strain)||5 preps||MN910A-1|
|MC-Easy Minicircle DNA Production Kit 10 rxn (does not include ZYCY10P3S2T producer E. coli strain)||10 preps||MN915A-1|
|MC-Easy Minicircle DNA Production Kit (Includes ZYCY10P3S2T producer E. coli strain)||5 preps||MN920A-1|
|MC-Easy Minicircle DNA Production Kit 10 rxn (Includes ZYCY10P3S2T producer E. coli strain)||10 preps||MN925A-1|
|MC-Easy Media Bonus Pack 5 rxn (includes Growth and Induction Media)||5 preps||MN950A-1|
Parental minicircle plasmids and the ZYCY10P3S2T Producer Bacterial Strain are available for purchase by not-for-profit researchers only. Commercial users may purchase pre-made mcDNA [ready-to-transfect] only. SBI also offers custom parental plasmid cloning and mcDNA production to both not-for-profit and commercial end users; contact firstname.lastname@example.org for additional details. For any other purposes, including the ability to buy the parental MC production system, commercial users should contact Stratech Scientific at email@example.com for further information.
Sustained expression after transfection
Minicircles are easier to transfect into most cell types and expression can persist for weeks. Below is an example of transfection of 1 ug minicircle DNA of the pMC.CMV-MCS-EF1-GFP-SV40PolyA (catalog # MN511A-1) construct using standard transfection protocols with HEK293 cells.
Minicircle Hydrodynamic transfection through tail vein injection
Minicircle DNA can be transfected into animal models through tail vein injection procedures. Expression of the transgene cassette is sustained for many months where typical plasmid DNAs are silenced within hours. Hydrodynamic tail vein injection of 40 ug minicircle DNA results in a slight drop after 48 hours but then stabilizes over time for persistent expression. Mix 40 ug minicircle DNA expression DNA in saline equal to 10% of the rodent’s body-weight (2 ml for a 20 g mouse). Inject into the tail vein rapidly (5-8 seconds).
Minicircle DNA Vector Options
SBI has created a set of minicircle parental cloning vector formats to choose from. Select from CMV or EF1a promoters, GFP, RFP and Puro markers. The minicircle parental cloning shRNA vector formats feature either CMV or EF1a expressing the markers and a downstream H1 promoter to express high levels of the cloned shRNA sequence. See the Table below for the particular MCS cloning sites for each of the minicircle DNA vectors.
MC-Easy™ Minicircle DNA production kit
The MC-Easy system enables the simple, reproducible and efficient way to produce high quality Minicircle DNA for your experiments. The finely tuned growth and induction media produce minicircle DNA that is free of parental and genomic DNA contamination. The Kit also includes an additional, innovative method for degrading any contaminating genomic DNA using an ATP-dependent DNase reagent that will selectively remove linear genomic and residual parental DNA but will not affect the minicircle DNA. Clean and effective minicircles every time. The system comes complete with a special minicircle DNA transfection reagent, MC-Fection, that works well with most cell lines. Choose from the minicircle production kit that includes the engineered E. coli producer strain ZYCY10P3S2T (cat# MN920A-1) or select the minicircle production and isolation kit alone (MN910A-1).
Kit components for catalog MN910A-1 and MN920A-1
5X Minicircle Growth medium (200 ml)
10X Induction Medium (100 ml)
Minicircle DNA Re-purification kit (5 preps)
Endotoxin free water (5 ml)
E. coli producer strain ZYCY10P3S2T (included in cat# MN920A-1)
Download the MC-Easy Minicircle DNA Production System Protocol
- Minicircle DNA Production System User Manual
Technical references and Product citations
- Mark A. Kay, Cheng-Yi He & Zhi-Ying Chen. A robust system for production of minicircle DNA vectors. Nature Biotechnology, (2010). doi:10.1038/nbt.1708. (PDF) »
- Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z, Panetta NJ, Chen ZY, Robbins RC, Kay MA, Longaker MT, Wu JC. A nonviral minicircle vector for deriving human iPS cells. Nature Methods. 2010 Mar;7(3):197-9.
- Osborn MJ, McElmurry RT, Lees CJ, Defeo AP, Chen ZY, Kay MA, Naldini L, Freeman G, Tolar J, Blazar BR. Minicircle DNA-based Gene Therapy Coupled With Immune Modulation Permits Long-term Expression of Î±-L-Iduronidase in Mice With Mucopolysaccharidosis Type I. Mol Ther. 2010 Nov 16.
- Huang M, Chen Z, Hu S, Jia F, Li Z, Hoyt G, Robbins RC, Kay MA, Wu JC. Novel minicircle vector for gene therapy in murine myocardial infarction. Circulation. 2009 Sep 15;120.
- Stenler S, Andersson A, Simonson OE, Lundin KE, Chen ZY, Kay MA, Smith CI, Sylvén C, Blomberg P. Gene transfer to mouse heart and skeletal muscles using a minicircle expressing human vascular endothelial growth factor. J Cardiovasc Pharmacol. 2009 Jan;53(1):18-23.
- Chen ZY, He CY, Ehrhardt A, Kay MA. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther. 2003 Sep;8(3):495-500.;