Experiment Summary

Genetic manipulation is a very powerful tool for studying diabetes, pancreatitis, and pancreatic cancer. Here we discuss the use of an adeno-associated virus (AAV) vector to modify gene expression, such as to introduce a green fluorescence protein (GFP) in wild-type mice, Cre recombinase in loxP mice, or to inactivate a gene with shRNA. The use of viruses for genetic modification allows for time-specific genetic changes which have advantages over time-consuming and often complex cross-breeding strategies. Here we provide a detailed approach for this process from viral production and purification through pancreatic ductal infusion. This protocol allows efficient delivery of AAV to mediate GFP or Cre expression for cell lineage tracing in the mouse pancreas or for the delivery of transgenes under a specific promoter to these cells.

Materials

A. Cell Culture and Triple Plasmid PEI Transfection

1. DMEM cell culture medium.
2. Opti-MEM reduced serum medium.
3. Fetal bovine serum (FBS).
4. T-150 flasks.
5. Sterile cell culture hood.
6. Cell culture incubator.
7. Countertop cell culture centrifuge with cooling system.
8. 1 mg/mL polyethyleneimine (PEI), pH 7.4.
9. Vector plasmid carrying the expression cassette flanked by viral ITRs, such as pAAV-CMV-ZsGreen, pAAV-Sox9-GFP, pAAV CMV-Cre, or pAAV-Sox9-Cre.
10. Packaging plasmid carrying the serotype 6 AAV rep and cap genes.
11. Helper plasmid carrying adenovirus helper proteins

B. Virus Harvesting and Purification

1. DMEM cell culture medium.
2. Benzonase.
3. 10% deoxycholate
4. Lysis buffer: 50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl₂, pH 8.0.
5. HEPES solution: 150 mM NaCl, 20 mM EDTA, 50 Mm HEPES, 1% Sodium lauroyl sarcosinate.
6. Chloroform.
7. PEG8000-NaCl solution: 40% polyethylene glycol (w/vol) in 2.5 M NaCl, average mol wt 8000.
8. PEG8000 solution: 40% polyethylene glycol (w/vol) in H20, average mol wt 8000.
9. 50% ammonium sulfate.
10. 50 mL conical tubes.
11. 1.5 and 2.0 mL microcentrifuge tubes.
12. Soft-walled 1.5 mL microcentrifuge tubes.
13. Plastic syringes.
14. 18G 25 mm needle.
15. 0.22 μM syringe filter.
16. Desktop centrifuge with cooling system.
17. 37°C water bath with shaker.
18. Orbital rotator.
19. Centrifugal filter unit with Ultracel-50K membrane.
20. Slide-A-Lyzer Dialysis Cassette, 10,000 MWCO.
21. AAVpro titration kit.

C. Pancreatic Ductal AAV Infusion

1. Infusion catheter.
2. Infusion syringe pump.
3. Plastic syringe.
4. 30G needle.
5. 31G blunt-ended cannula.
6. Nair hair removal cream.
7. Surgical tape.
8. 6-0 Silk suture with BV-1 needle.
9. 4-0 Vicryl suture with RB-1 needle.
10. 1.5 and 2.0 mL microcentrifuge tubes.
11. Cotton applicator Q-tips.
12. Dissecting microscope.
13. Dietrich micro bulldog clamp.
14. Olsen-Hegar combination scissor and needle holder.
15. Arruga micro forceps.
16. Hudson forceps.
17. Micro dissecting scissors.
18. Isoflurane.
19. Plexiglass heat pad.
20. Betadine.
21. 70% ethanol.
22. Ketoprofen.
23. Warm saline.
24. 8-Week-old C57BL/6J mice.
25. 8-Week-old ROSA-CAG-LSL-tdTomato reporter mice.

Note: (Pre-made AAV particles such as, Cre-GFP Adeno-associated virus(AAV Serotype 6), Cre-GFP Adeno-associated virus(AAV Serotype 8), CMV-Cre AAV (Serotype Retrograde), etc. can also be used in this experiment without transfection and purification steps.)

Procedure

A. Cell Culture and Triple Plasmid PEI Transfection

1. Maintain HEK293 cells in DMEM with 10% FBS.
2. When cells reach 80% confluency, change the culture medium to serum-free DMEM (10 mL for one T-150 flask) in the morning before transfection.
3. For transfection, mix the vector plasmid, packaging plasmid, helper plasmid, and PEI in Opti-MEM medium. Incubate 20 min at room temperature (RT). Aspirate medium from flasks. Add 10 mL of serum-free DMEM to each flask. Add 1 mL of transfection plasmids-PEI solution to each flask. Return the flasks to incubator.
4. The following day, add 1 mL of FBS to each flask. Return to incubator for 3 days.

B. Virus Harvesting and Purification

1. After 3 days, remove flasks from the incubator and, using a large cell scraper, remove cells from surface. Using a 10 mL pipette, wash down the flask to ensure all cells are removed. Collect cells with medium and transfer to a 50 mL conical tube.
2. Approximately 250 mL of culture medium from twenty T-150 flasks will be collected. Centrifuge the culture medium with cells at 3000 × g for 10 min to pellet the cells and cellular debris.
3. Pour the supernatant into seven clean 50 mL conical tubes. Distribute the supernatant so that six tubes have 40 mL and one tube has about 10 mL.
4. Add 0.5 mL of lysis buffer to the cell pellets, then combine the pellets and buffer into one of the tubes.
5. Place the cell pellet suspension tube in a -80°C freezer and store the culture medium supernatant tubes in a 4°C refrigerator.
6. Once frozen, remove the cell pellet suspension from the freezer and thaw in a 37°C water bath. Repeat for a total of three freeze/thaw cycles.
7. After the third thaw cycle, add 50 U/mL Benzonase and incubate at 37°C for 30 min with shaking.
8. Add deoxycholate to a concentration of 1% and again incubate at 37°C for 45 min with shaking.
9. Transfer the contents of the 50 mL cell suspension tube to multiple 2.0 mL microcentrifuge tubes. Spin in a microcentrifuge at 14,000 rpm (17,000 × g) for 10 min at RT.
10. Transfer the supernatant from each cell suspension tube into a single clean 50 mL tube and measure volume. Discard the pellets and microcentrifuge tubes.
11. Remove the 7 culture medium supernatant tubes from 4°C and place on ice. Add an equal volume of the cell suspension supernatant to each culture medium supernatant tube.
12. Add 10 mL of PEG8000-NaCl solution to the tubes containing 40 mL of supernatant from step 3. Add 2.5 mL of PEG8000-NaCl solution to the tube containing only 10 mL of supernatant from step 3. Incubate the tubes at 4°C on an orbital rotator for 45 min.
13. Remove tubes from the cold room and spin in a cell culture countertop centrifuge at 1500 × g for 5 min at 4°C.
14. Aspirate supernatant. Add 0.5 mL of HEPES solution to the pellets. Mix thoroughly with a pipette to detach pellets, and consolidate all solution and pellets into a single 50 mL tube.
15. Vortex the tube for 1 min to break up pellets. Measure the volume with a pipette.
16. Add an equal volume of chloroform to the tube and vortex for 2 min.
17. Transfer the viral solution, which should appear like milk, to microcentrifuge tubes and spin in a microcentrifuge at 14,000 rpm (17,000 × g) for 10 min at RT.
18. After centrifugation, carefully transfer the supernatant to a 50 mL tube and add the recommended volume of premixed 40% PEG8000 solution (prewarmed at room temperature) and 50% ammonium sulfate.
19. Vortex vigorously for 2 min, then transfer the mixture equally to soft-walled 1.5 mL microcentrifuge tubes and spin in a microcentrifuge at 14,000 rpm (17,000 × g) for 10 min at RT.
20. Withdraw the clear bottom aqua phase containing the virus with an 18G needle and syringe, and transfer to a 50 mL conical tube.
21. Transfer the aqueous phase product to a Slide-a-Lyzer 10,000 MWCO dialysis cassette. Dialyze the aqueous phase product against 1× PBS at RT to remove salt for 4 h. Change PBS buffer and dialyze for another 4 h.
22. Transfer the dialysis cassette to DMEM and place on a rotator for 2 h.
23. Concentrate the virus.
24. Titration of AAV can be performed using an AAVpro qPCR titration kit and following the manufacturer’s instructions.

C. Pancreatic Ductal AAV Infusion

1. Prepare approximately 100 μl per mouse of ~1 × 10¹² vector genomes (vg) of AAV in DMEM with phenol red.
2. Set up infusion equipment as shown in Fig. 1.

Fig. 1 Organization of the equipment for mouse pancreatic ductal infusion.

3. Anesthetize mice with 2-2.5% (w/vol) isoflurane by inhalation.

4. Immobilize the mouse on the plexiglass heat pad using surgical tape, and remove hair. Clean and disinfect the now hairless area with gauze soaked in 70% ethanol.

5. Laparotomy: Use scissors to incise the skin at the midline of the abdomen. Next, cut the abdominal muscle along the linea alba to create a midline upper abdominal laparotomy incision of 1.5 cm.

6. Exposure of the pancreatic duct using Q-tips to gently bring the stomach into the incision. Rotate and stretch the duodenum to expose the biliary-pancreatic duct and its junction within the duodenum (the sphincter of Oddi), which appears pale.

7. Perform a slight backward rotation of the duodenum to expose the distal biliary-pancreatic duct. With a view of the back side of the duodenum and the duodenal portion of the pancreas, the region of the papilla should also be visible.

8. To prevent perfusion of the liver, place a micro bulldog clamp on the common bile duct above the branching of the pancreatic duct. Avoid clamping the portal vein, located behind the common bile duct, while performing this step.

9. Use a 30G needle to make a small hole in the duodenum opposite to the sphincter of Oddi, 1-2 mm away from the ampulla of Vater. Next, pass a 31G blunt-ended cannula through this small hole in the duodenum and then the sphincter of Oddi (Fig. 2a). The tip of the catheter should be positioned at the origin of the pancreatic duct branch in the biliarypancreatic duct (Fig. 2a).

10. Clamp the ampulla around the catheter with another micro bulldog clamp to prevent backflow or leakage into the duodenum during the viral infusion (Fig. 2b). Connect the back end of the catheter to the infusion syringe pump.

Fig. 2 Detailed positioning of pancreatic ductal infusion.

11. Turn on the pump and infuse the predetermined amount of solution.

12. After the infusion is complete, remove the micro bulldog clamp on the sphincter of Oddi and gently withdraw the catheter from the duct and out of the duodenum. Next, remove the micro bulldog clamp from the biliary-pancreatic duct.

13. Close the hole.

14. Return the intestines to the abdomen and suture the abdominal wall closed with a 4-0 Vicryl suture using a running stitch.

15. Return the mouse to its cage on a heated pad until it fully recovers. Give ketoprofen at a dose of 5 mg/kg subcutaneously once per day for 3 days after surgery for analgesia.

16. You should see demonstrable results from the procedure approximately one week after viral infusion. Euthanize the animal and harvest the pancreas at the desired experimental endpoint. If using a virus with fluorescent proteins, fluorescence can be visualized under a stereo dissecting microscope (Fig. 3) or with whole mount staining (Fig. 3c, f).

Fig. 3 Visualized mouse pancreatic tissues.