Generating and Amplifying Adenoviral Constructs: Difference between revisions

(2 intermediate revisions by 2 users not shown)

Line 12:

*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==

Line 27:

Line 30:

#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.

===desalting of CsCl purified adenovirus using PD-10 desalting columns (17-0851-01 GE Healthcare)===

# Cut column, discard storage solution and wash X5 with ~5ml PBS

# Add virus sample (up to 2.5 ml)

# Elute with 5 ml PBS, discard first 20 drops and collect 10 drop fractions into 10 tubes.

# OD 260 fractions in nanodrop, virus is in middle fractions (5-7 in my extraction). Good virus yield is >3 OD.

# dilute to 1 OD/ml

# Add 10% glycerol and store at -80.

===Tail Vein Injection===

# Insert mouse to restriction device. Carefully warm tail with lamp/warm water, inject 0.1 OD/mouse (0.15 for HFD mouse) using insulin syringe.

# Injected mice are housed in biohazard mouse room for 3 days (need bright-pink barcode stickers and water bottles).

# Adenovirus is detected in liver for at least 2-3 weeks, shRNA has maximal effect at 4-6 days, usually gone in 10 days.

@@ Line 12: / Line 12: @@
 *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==
@@ Line 27: / Line 30: @@
 #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.
+===desalting of CsCl purified adenovirus using PD-10 desalting columns (17-0851-01 GE Healthcare)===
+# Cut column, discard storage solution and wash X5 with ~5ml PBS
+# Add virus sample (up to 2.5 ml)
+# Elute with 5 ml PBS, discard first 20 drops and collect 10 drop fractions into 10 tubes.
+# OD 260 fractions in nanodrop, virus is in middle fractions (5-7 in my extraction). Good virus yield is >3 OD.
+# dilute to 1 OD/ml
+# Add 10% glycerol and store at -80.
+===Tail Vein Injection===
+# Insert mouse to restriction device. Carefully warm tail with lamp/warm water, inject 0.1 OD/mouse (0.15 for HFD mouse) using insulin syringe.
+# Injected mice are housed in biohazard mouse room for 3 days (need bright-pink barcode stickers and water bottles).
+# Adenovirus is detected in liver for at least 2-3 weeks, shRNA has maximal effect at 4-6 days, usually gone in 10 days.