Abstract
Vi konstruerade linjära kovalent slutna (LCC) DNA minivectors som en icke-viral gen-leverans vektor alternativ som produceras via en enkel plattform in vivo. DNA ministrings har en ökad säkerhetsprofil och även effektivt leverera DNA last till målceller. Konventionella DNA-vektorer bär oönskade prokaryota sekvenser, inklusive antibiotikaresistensgener, CpG-motiv, och bakteriella replikations, vilket kan leda till stimulering av värdimmunologiska reaktioner. Biotillgängligheten av konventionella DNA-vektorer är också äventyras på grund av deras större molekylstorlek. Deras cirkulär natur kan också ge kromosomal integration, vilket leder till insertionsmutationer.
Bakteriella sekvenser skärs ut från DNA minivectors, vilket innebär att endast den intressanta genen (GOI) och nödvändiga eukaryota uttryckselement. Våra LCC DNA minivectors eller DNA ministrings, saknar immunogena bakteriella sekvenser; därför förbättra thEIR biotillgänglighet och GOI uttryck. I händelse av vektorn integreras i kromosomen, kommer LCC DNA ministring letalt störa värdkromosomen, varigenom avlägsnande av potentiellt farligt mutanten från prolifererande cellpopulationen. Följaktligen DNA ministrings erbjuder fördelarna med "minicircle" DNA samtidigt som man eliminerar risken för oönskade händelser vektor integration. I jämförelse med konventionella plasmider och deras isogena cirkulära kovalent slutna (CCC) motsvarigheter, DNA ministrings ingen signifikant bättre biotillgänglighet, transfektionseffektiviteten, och cytoplasmatiska kinetik - de kräver sålunda mindre mängder av katjoniska ytaktiva medel för effektiv transfektion av målceller.
Vi har konstruerat en en-stegs inducerbar in vivo-system för produktion av DNA-ministrings i Escherichia coli som är enkel att använda, snabb, och skalbar.
Materials
| Name | Company | Catalog Number | Comments |
| E. coli W3NN | Mediphage | strain for ministring processing: Nafissi & Slavcev, 2012 | |
| pDMS.IRES.GFP | Mediphage | parent plasmid for ministring processing: also referred to as pDNA Ministring (pDMS) | |
| BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani) | Fisher Scientific | 244620 | |
| Fisher BioReagents Terrific Broth | Fisher Scientific | BP2468500 | |
| BD Bacto Dehydrated Agar | Fisher Scientific | 214010 | |
| Ampicilin | MP Biomedicals LCC | 2190147 | |
| Ultrapure Milipore Water | Fisher Scientific | Z00Q0V0US | Model: Mili-Q Advantage A10 Water Purification System |
| Surgical Gloves | VWR | (S) 82026-424, (M)82026-426, (L) 82026-428, (XL) 82026-430 | |
| Fisherbrand Petri Dishes with Clear Lid Raised Ridge (100 x 15 mm) | Fisher Scientific | FB0875712 | |
| 30 ml KIMAX Test Tubes | VWR | 89001-448 | |
| Spectrophotometer Cuvette | Sarstedt | 67.74 | |
| Sterile 15 ml Falcon Tubes | BD Falcon | 62406-200, | |
| Sterile 50 ml Centrifuge Tubes | FroggaBio | 2930-S0 | |
| Falcon Disposable Polystyrene Serological Pipets 10 ml | BD Falcon | 13-675-20 | |
| Falcon Disposable Polystyrene Serological Pipets 25 ml | BD Falcon | 13-668-2 | |
| Micropipettor (100-1,000 μl) | FroggaBio | C1000-1 | |
| Micropipettor (20-200 μl) | FroggaBio | C200-1 | |
| Sterile Pipette Tips (100-1,250 μl) | VWR | 89079-472 | |
| Sterile Pipette Tips (1-200 μl) | VWR | 89079-460 | |
| Fisher Scientific Economy Burner | Fisher Scientific | 03-917Q | |
| Incubator Shaker | New Brunswick Scientific | M1352-00000 | |
| Sterile Erlenmeyer Flask (125 ml) | Pyrex (Sigma Aldrich) | CLS4980125 Aldrich | |
| Sterile Erlenmeyer Flask (250 ml) | Pyrex (Sigma Aldrich) | CLS4980250 Aldrich | |
| Sterile Erlenmeyer Flask (500 ml) | Pyrex (Sigma Aldrich) | CLS4980500 Aldrich | |
| Spectrophotometer | Thermo Fisher Scientific | 335901 | Model: Genesys 10 Vis |
| Floor Centrifuge | Beckman Coulter | 369001 | Model: Avanti J-E |
| Benchtop Centrifuge | Beckman | 362114 | Model: GS-6R |
| Fisher Scientific Analog Vortex Mixer | Fisher Scientific | 02-215-365 | |
| 1 kb DNA Ladder | New England BioLabs | N3232S |
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