Lentivirus Production and Purification

Fig. 1

GFP expression following transduction with a VSV-G pseudotyped LV vector. LV vectors produced using the current protocol efficiently transduces cells in vitro (a–c) and in vivo (d). SH-SY5Y (a), MN9D (b), or PC12 (c) cells were plated and grown to approximately 50 % confluency. Cells were transduced with the VSV-G pseudotyped LV vector expressing GFP under the control of the cytomegalovirus/chicken β-actin hybrid promoter at a multiplicity of infection (MOI) of 100. GFP expression was visualized 48 h post-transduction. To demonstrate LV vector transduction in vivo, adult male rats were injected with 2 μL of the VSV-G pseudotyped LV vector expressing GFP under the control of the cytomegalovirus/chicken β-actin hybrid promoter (2.07 × 10¹² vg/mL) into the striatum. Animals were sacrificed 4 weeks post-transduction, and native GFP fluorescence was visualized in the striatum (d). The inset in panel (d) shows a higher magnification image of the area within the box in panel (d). Scale bar in panel (a)–(c) represents 50 μm. Scale bar in panel (d) and inset represent 200 μm and 50 μm, respectively

2 Materials

2.1 Plasmids and Cells

Human Embryonic Kidney 293 T (HEK-293 T) Cells (ATCC, Manassas, VA, USA).

pNHP packaging vector (Addgene, Cambridge, MA, USA; see Notes 1 and 2 ).

pHEF-VSVG (Addgene, Cambridge, MA, USA; see Note 3 ).

pFIN transfer vector (Addgene, Cambridge, MA, USA; see Note 4 ).

2.2 Reagents and Supplies

Penicillin/Streptomycin (Pen/Strep; 10,000 units/mL).

Fetal bovine serum (FBS).

TrypLE Express 1× dissociation reagent (Thermo Fisher Scientific, Grand Island, NY, USA).

25 kDa linear Polyethylenimine (PEI) (Polysciences, Warrington, PA, USA).

Dulbecco’s modified Eagle media (DMEM) containing high glucose, glutamine, and sodium pyruvate.

1× Dulbecco’s phosphate-buffered saline (DPBS) containing calcium and magnesium.

0.22 μm Stericup filter unit (EMD Millipore, Billerica, MA, USA).

1.5 M NaCl: Add 87.66 g of NaCl to 800 mL of dH₂O, stir until completely dissolved. Adjust to 1 L with dH₂O and filter sterilize with a 0.22 μm filter unit.

0.45 μm Stericup-HV filter units (EMD Millipore, Billerica, MA, USA).

10.

Centricon Plus-70 Centrifugal filter units (EMD Millipore, Billerica, MA, USA).

11.

38.5 mL conical thinwall polyallomer ultracentrifuge tubes or equivalent (Beckman Coulter, Indianapolis, IN, USA).

12.

T175 Culture flasks.

13.

SW32Ti rotor (Beckman Coulter, Indianapolis, IN, USA) or equivalent.

14.

Beckman Coulter L-100XP Ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA) or equivalent.

15.

Sorvall RC 6 superspeed centrifuge (Thermo Fisher Scientific, Grand Island, NY, USA) or equivalent.

16.

Real-time PCR system: Applied biosystems 7500 (Thermo Fisher Scientific, Grand Island, NY, USA) or equivalent.

17.

Lenti-X-qRT-PCR kit (Clonetech, Mountain View, CA, USA).

18.

Standard tissue culture equipment.

3 Methods

3.1 Prepare Reagents

Prepare complete HEK-293 T media (DMEM with 10 % FBS and 1 % Pen/strep). Add 100 mL of FBS and 10 mL of Pen/strep to 890 mL of DMEM (see Note 5 ).

Prepare PEI transfection reagent. Heat 200 mL of dH₂O to 80 °C in a beaker on a stirring hotplate. Add 80.75 mg of PEI to the beaker and stir with a stir bar. Stir at 80 °C until the PEI is completely dissolved. Adjust pH to 8.0 with hydrochloric acid. Adjust to 250 mL with dH₂O. Filter sterilize with a 0.22 μm filter unit (see Note 6 ).

Prepare the required amount of plasmid DNA (for each respective plasmid) using a standard plasmid DNA preparation method. DNA must be endotoxin free.

3.2 Transfection and Viral Purification

Seed the T175 flask with HEK-293 T cells. Grow HEK-293 T cells in a T75 starter flask prior to seeding the T175. Once the T75 starter flask is at 90–95 % confluency aspirate media from the flask and gently rinse with sterile PBS. Incubate cells with 4 mL of dissociation reagent (e.g., TrypLE Express) at 37 °C for 5 min. Add 6 mL of media containing serum to deactivate the dissociation reagent, and triturate with 25–30 full strokes of a serological pipette to create a single cell suspension.

Determine cell number per mL using a hemocytometer.

Calculate the amount of the single cell suspension needed to seed the T175 flask with 3 × 10⁷ HEK-293 T cells. Add cells to flask and bring the total volume of the flask to 28 mL with warm (37 °C), complete HEK-293 T media.

Incubate T175 Flask at 37 °C with 5 % CO₂ overnight or until 80–90 % confluency is reached.

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