Experiment Summary

Recombinant viral vectors have been used to study a variety of fundamental issues in developmental neurobiology, as well as pathogenesis and treatments for various neurodegenerative diseases. Lentiviral vectors are valuable tools for neurobiology research, because of their ability to transduce nondividing cells such as neurons, to introduce new genes into central nervous system (CNS) cells in vivo and in vitro.

Reagents

1. 293FT cells
2. Anesthesia
3. CaCl2 (2 M; filter-sterilized)
4. Culture medium for retroviral vector production
5. Phosphate-buffered saline
6. Ethanol (70%)
7. HEBS (2×)
8. Lipofectamine 2000
9. Mice (C57Bl/6, female, 6 -10 week old)
10. Opti-MEM I, reduced serum medium
11. Puralube vet ointment
12. Sucrose (20%)
13. Prepare in PBS and filter sterilize
14. Tissue adhesive (3M Vetbond)
15. Viral DNA mix (select one):

Viral DNA mix for lentiviral vectors

Viral DNA mix for MML retroviral vectors

Equipment

1. Biosafety level-2 facility
2. Centrifuge
3. Conical tubes, 50-mL
4. Culture plates
5. Drill bur, size no. 1
6. Electric drill
7. Electric hair trimmer or small scissors
8. Filtration units, 0.45-μm
9. Fluorescence microscope
10. Marker pen
11. Microsyringes
12. Needles for microsyringes, 33-gauge
13. Polyallomer tubes, 13 × 51 mm
14. Polyallomer tubes, 25 × 89 mm
15. Rotors for ultracentrifuge SW32 Ti or SW28, SW55 Ti
16. Stereotaxic frame for small animals
17. Surgical instruments, sterile
18. Tissue culture incubator, set at 37°C and 5% CO₂
19. Tissue culture plates, 24-well
20. Tubes, 0.5-mL, sterile
21. Tubes, 14-mL, polypropylene
22. Ultracentrifuge
23. Warming blanket and mouse cage

Procedure A: Transfection with Lipofectamine 2000

Day 1 (30 min)

1. Passage 293FT cells into twelve 10-cm cell culture plates at 5 × 10⁶ cells per plate. Use 10 mL of complete medium without antibiotics per plate.

Day 2 (6 h)

2. Add lentiviral or retroviral DNA mix and 2.4 mL of Opti-MEM into four 14-mL polypropylene tubes (A tubes). Mix by tapping each tube.

3. Add 2.4 mL of Opti-MEM and 150 µL of Lipofectamine 2000 into four additional 14-mL polypropylene tubes (B tubes). Mix by tapping each tube. Incubate at room temperature (20°C-25°C) for no longer than 5 min.

4. Pipette the contents of one B tube into one A tube. Mix by inverting the tubes. This results in four tubes of 4.8-mL DNA/lipofectamine mix.

5. Incubate the tubes at room temperature for 25-30 min.

6. Add 1.6 mL of the DNA/lipofectamine mix dropwise onto each plate of 293FT cells. The drops

should evenly cover the surface of the culture medium. Mix by gently rocking the plate.

7. Incubate the cells for 5 h at 37°C and 5% CO₂.

8. Replace the supernatant with 10 mL of fresh complete medium containing antibiotics.

9. Incubate the plates for 48 h until day 4.

Day 3 (10 min)

10. Check GFP fluorescence to evaluate the transfection efficiency. Proceed to Step 21.

Procedure B: Transfection with Calcium Phosphate Precipitation

Day 1 (1 h)

11. Passage 293FT cells into sixteen 15-cm cell culture plates at 10 × 106 cells per plate. Use 15 mL of complete medium with antibiotics per plate.

Day 2 Late Afternoon (30 min)

12. Add lentiviral or retroviral DNA mix (x mL) and sterile ddH2O (5.1 - x mL) into two 50 mL polypropylene tubes. Mix by tapping the tubes.

13. Add 2.9 mL of CaCl₂ solution to each tube. Mix by pipetting.

14. Add 8 mL of 2× HEBS solution dropwise into the DNA/CaCl₂ tubes.

15. Mix by pipetting and then by bubbling air from the pipette for 30 sec.

16. Incubate for 10 min at room temperature.

17. Add 2 mL of the mix dropwise onto each plate of 293FT cells. The drops should evenly cover the surface of the culture medium. Mix by gently rocking the plate.

18. Incubate the plates overnight at 37°C and 5% CO₂.

Day 3 Morning (10 min)

19. Check GFP fluorescence to evaluate transfection efficiency.

20. Replace the medium with 15 mL of fresh complete medium with antibiotics.

Concentration of Viral Vectors

Day 4 (20 min)

21. Collect the medium containing viral vectors into sterile containers. Provide each plate of cells with 10 (Procedure A) or 15 (Procedure B) mL of fresh complete medium with antibiotics.

22. Store the collected medium at 4°C.

Day 5 (8-10 h)

23. Collect the medium containing viral particles into sterile containers.

24. Divide the collected medium from days 4 and 5 into 50 mL conical tubes. To remove cell debris, centrifuge the tubes at 1000 g for 3 min.

25. Filter the supernatant through a 0.45-μm filter unit.

26. Transfer the filtered supernatant into 25 × 89-mm ultracentrifuge tubes (A tubes).

27. Centrifuge the filtered supernatant in an SW32 Ti rotor at 65,000g for 2 h at 4°C.

28. Aspirate off the supernatant.

29. Add PBS (0.5 mL for the lipofectamine method or 0.25 mL for the calcium phosphate method) to the precipitated viral vectors into each A tube. Resuspend the precipitate by pipetting it up and down 20 times. Pool the resuspended viral vector in PBS from all the A tubes into a single 4-mL 13 × 51-mm ultracentrifuge tube (B tube).

30. Sequentially wash the A tubes with 0.5 mL of PBS, and transfer the wash into the 4-mL B tube.

31. (Optional) Add 0.5 mL of a 20% sucrose cushion (made in PBS and filter sterilized) to the B tube.

32. Centrifuge the B tube in an SW55 Ti rotor at 65,000g for 2 h at 4°C.

33. Aspirate off the supernatant.

34. Resuspend the final pellet in 80 µL of PBS by vortexing for 30 sec and then by pipetting 20 times. This procedure is enough to resuspend the virus. Leave the remaining sticky pellet, and transfer the suspended viral vector solution to a sterile 0.5-mL tube (C tube).

35. Wash the B tube once with 20 µL of PBS, and transfer the wash to the C tube.

36. Briefly centrifuge the C tube, and transfer the supernatant to a new 0.5-μL tube.

37. Aliquot the viral vectors into 5-10 µL portions, and store them at -80˚C.

Determine Virus Titer

38. Seed 1 × 105 293FT cells per well in a 24-well plate, and incubate the cells overnight at 37°C and 5% CO₂.

39. Perform a serial dilution (e.g., 10⁴, 10⁵, 10⁶, and 10⁷ for CAG promoter retroviral vectors;10⁵, 10⁶, 10⁷, and 10⁸ for CMV promoter lentiviral vectors).

40. Transfer 10 µL of each dilution to individual wells.

41. Three to 5 d later, view the infected cells with a fluorescent microscope, and count the number of fluorescent clusters. Calculate the number of colony-forming units per milliliter (cfu/mL) of original virus solution.

Injection of Viral Vectors into the Brain

42. Deeply anesthetize mice.

43. Shave a small area on the head with an electric trimmer. Apply eye ointment to prevent the eyes from drying out too much.

44. Make a 1-cm incision in the skin over the skull within the shaved patch. Clean the incision of blood.

45. Mount the mouse onto a stereotaxic frame.

46. Move the tip of the injection needle to the bregma.

47. Move the needle tip according to the lateral and posterior coordinates from the bregma.

48. Mark the position with a marker pen.

49. Move the needle out, and using an electric drill, make a small hole in the skull at the marked site.

50. Dip the needle tip into the virus solution (from Step 37), and load 1.5 µL of solution into the syringe.

51. Move the needle tip to the hole in the skull, and set it level with the surface of the skull.

52. Move the needle tip down to a predetermined dorsoventral coordinate from the level of the skull surface.

53. Inject a total of 1.5 µL of virus solution.

54. After finishing the injection, wait for 1 min to prevent the injected solution from flowing back through the needle track.

55. Move the needle tip up 1 mm, and then wait for another 1 min. Slowly retract needle from mouse brain.

56. Rinse the needle first with 70% ethanol and then with PBS.

57. Close the skin using tissue glue, or suture it with thread.

58. Place the mouse in a cage on top of a warm blanket until it is fully alert. Transfer the mouse back to its home cage.

59. After a certain survival period (minimum of 3 d for GFP expression), analyze the properties of interest using suitable methodologies.