Generating and Amplifying Adenoviral Constructs: Difference between revisions

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 *100% ethanol
 *2 mm electroporation cuvette
+*25 cm2 and 75 cm2 tissue culture flasks.
+*Ultraclear pollyallomer tubes for Ti45 rotor (Beckman Cat# 344087)
+*2x Virus storage buffer (10 mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA and 50% glycerol; filter sterilized)
 ==Protocol==
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 #Add 8 uL resuspended linearized plasmid to 20 uL electrocompetent cells
 #Carefully transfer to ice cold 2 mm electroporation cuvette avoiding bubbles and keeping on ice
-#Electroporate at '''pulse at 2,500 V, 200 O and 25 mF'''
+#Electroporate at '''2,500 V, 200 O and 25 mF'''
 #Resuspend in 500 uL LB and plate on 2-5 LB/Km plates
 #Pick 10-20 of the smallest colonies the next day and grow in LB/Km broth
 #Miniprep clones.  Digest 10 uL with PacI and check size by running out both digested and undigested clones on a 0.8% agarose gel.  Correct recombinants usually yield a large fragment (approximately 30 kb) and a smaller fragment of 3.0 or 4.5 kb.
 #Retransform and amplify correct clone(s).
+===Transformation into 293A Cells===
+#Plate cells so that at time of transformation they are 50-70% confluent.
+#Digest recombinant plasmid with PacI in 100 uL with 3ug plasmid and 100U PacI.
+#Precipitate with 70% ethanol, dry and resuspend in 20 uL sterile water.
+#Mix 3 ug PacI digested plasmid and 15 uL LipofectAMINE reagent for each 25-cm2 tissue culture flask in 500 uL Opti-MEM I, and incubate the DNA/LipofectAMINE reagent mix for 15–30 min at room temperature.
+#While waiting, wash cells with serum free DMEM and add OptiMEM (5 mL/10 cm dish)
+#Add DNA/Lipofectamine and incubate 4-6h.
+#Replace media with complete DMEM
+#The next day check transformation efficiency by GFP fluoresence (for pAdtrack)
+#Do not remove media, but add 2 mL fresh complete DMEM every 5-7 days.  Wait 2-3 weeks before collecting viral particles.
+===Preparation and Purification of Viral Particles===
+#Scrape cells into 15 mL conical tube.
+#Aspirate all but the final 2 mL of cells and resuspend by vortexing
+#Release viral particles with four freeze-thaw cycles (liquid nitrogen then 37C water bath)
+#Centrifuge at 500g at 4C to pellet debris.
+#Store supernatant (virus) at -80 or use immediately to amplify
+===Amplification of Adenovirus===
+#Plate 293A cells in 25-cm2 tissue culture flasks at 80–90% confluency in 7 ml complete DMEM 6–15 h before infection.
+#Infect 293A cells by adding 40–50% of the primary transfection viral supernatants (i.e., 0.5–1.0 ml of the 2.0 ml viral lysate) to each 25-cm2 flask.
+#Check transduction by GFP fluorsesence (for pAdtrack)
+#Collect cells by scraping into a 15 mL conical 3-5 days post-infection.
+#Remove all but 5 mL media by aspiration.
+#Freeze/Thaw cells 4 times as described above
+#Centrifuge at 500g to pellet debris
+#Store supernatant (virus) at -80 or use immediately to amplify again.
+#Repeat amplification three time more first with one 75 cm2 flask (using entire supernatant) then with 3-5 75 cm2 flasks, then with 10-20 75cm2 flasks using entire viral supernatant as the innoculant each time.
+#For final amplification resuspend pelleted cells in 8 mL D-PBS -/-, perform 4 freeze thaw cycles and centrifuge 10 min at 7000g
+===Purification of Adenovirus===
+#Transfer supernatant to 50 mL conical tube and add 4.8g of Cesium chloride and dissolve
+#Transfer 10 mL to 12 mL polyallomer tubes (for SW 45-Ti) add ~ 2mL mineral oil to top of tube to prevent tube crushing.
+#Centrifuge 18–24 h at 176,000g (SW 41 Ti rotor at 32,000 r.p.m.)
+#The purified virus should be 1-2cm below the mineral oil in an opaque layer.
+#Remove virus layer to a clean falcon tube using a syringe (do this in a beaker to easier add bleach to waste).
+#Add an equal volume of 2X Virus storage buffer and store at -80
+#Titer the virus using a kit.