Produktion av dubbel DNA Ministrings

1School of Pharmacy, University of Waterloo, 2Sgd Health Innovation
Published 2/29/2016
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Wong, S., Lam, P., Nafissi, N., Denniss, S., Slavcev, R. Production of Double-stranded DNA Ministrings. J. Vis. Exp. (108), e53177, doi:10.3791/53177 (2016).

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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

DOWNLOAD MATERIALS LIST

References

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