Generation of Recombinant Lentivirus in Mammalian Cells

In This Article

Abstract

Source: Zhang, Y., et al., Direct Reprogramming of Mouse Fibroblasts into Melanocytes. J. Vis. Exp. (2021)

This video demonstrates the production and concentration of recombinant lentivirus in mammalian cells, enabling efficient gene delivery of transcription factors or other genetic elements for applications such as cellular reprogramming, functional studies, and potential therapeutic research.

Protocol

1. Construction of a concentrated lentivirus packaging system for transcription factors

Production of the concentrated virus (Figure 1A, B)
1. Plate 1.5 × 10⁶ HEK (human embryonic kidney)-293T cells into a 60 mm dish and culture these cells with normal medium (see Table 1) at 37 °C in a humidified incubator with 5% CO₂.
  NOTE: If the virus needs to be packaged in batches, a 100 mm cell culture dish can be used (for details, see Table 2).
2. After 24 h, ensure that the HEK-293T cells have reached 80-90% confluence on the day of transduction, and replace the medium with 3.5 mL of DMEM (Dulbecco’s modified eagle medium) (150 µL of DMEM per 1 cm²). Leave the cells at 37 °C in a humidified incubator with 5% CO₂ for 2 h.
  NOTE: The replacement medium must be serum-free, so that the cells can be "starved" for better plasmid transfection.
3. Prepare the mixture of plasmids (mix A) containing 3 µg of the target plasmids of Mitf, Sox10, Pax3, Sox2, Sox9, or Snai2; 1 µg of the packaging plasmid PMD2.G, and 2 µg of the packaging plasmid PSPAX2. Make up the volume of mix A to 150 µL with serum-free DMEM. Prepare the mix of the transfection reagent (mix B) by adding 12 µL of the transfection reagent (the volume is twice the total mass of all the plasmids), and make up the volume of mix B to 150 µL with serum-free DMEM.
  NOTE: When preparing the mixes, it is important to add liquid slowly to avoid air bubbles.
4. Combine mix A and mix B after allowing them to stand for 5 min at room temperature. Incubate the mixture at room temperature for 20-30 min to form the transfection complex.
5. Take the HEK-293T cells out of the incubator, replace the medium with DMEM + 2% FBS (fetal bovine serum), add the mixture from step 1.1.4 dropwise, and mix the liquid gently.
6. After 8 h, change the medium with 3.5 mL of normal medium. After changing the medium, collect the virus supernatant every 24 h and 48 h.
7. Mix the virus supernatants collected at two different time points. Centrifuge at 200 × g for 5 min at 4 °C. Pass the supernatant through 0.45 µm filters and collect it in a 50 mL sterile conical tube.
  NOTE: The virus collected at 24 h can be stored at 4 °C and mixed with the virus collected at 48 h.
8. Concentrate the virus supernatant by centrifuging at 6000 × g at 4 °C overnight (~16 h). Ensure that the virus pellet is visible at the bottom of the conical tube after centrifuging.
9. Pour out the supernatant slowly. Dissolve the virus pellet in a volume of normal medium that is 1/100^th of the volume of the virus supernatant. Using a P1000 micropipette, pipette up and down gently until a homogeneous mixture is obtained. Divide the concentrated virus into microcentrifuge tubes as needed. Store at −80 °C.
  NOTE: The virus is concentrated 100x with this method. The concentrated virus (100x) can be stored for >1 year at −80 °C. Avoid repeated freezing and thawing of the virus.

Table 1: Components of normal and reprogramming media.

	Components	Dose (concentration, volume)	Final Concentration
Normal Medium (100 mL)	DMEM/HIGH GLUCOSE	89 mL
	Heat-inactivated FBS	10 mL
	Antibiotics(Pen/Strep)	10000 U/mL, 1 mL	100 U/mL
Reprogramming medium (100 mL)	RPMI-1640	88 mL
	Heat-inactivated FBS	10 mL
	Recombinant Human SCF	200 μg/mL, 50 μL	100 ng/mL
	Recombinant Human bFGF	4 μg/mL, 250 μL	10 ng/mL
	Recombinant Human insulin	10 mg/mL, 50 μL	5 μg/mL
	EDN3 human	100 μM, 100 μL	0.1 μM
	Cholera Toxin	0.3 mg/mL, 0.56 μL	20 pM
	Phorbol 12-myristate 13-acetate (TPA)	1 mM, 20 μL	200 nM
	Hydrocortisone	100 μg/mL, 500 μL	0.5 μg/mL
	Adenine	40 mg/mL, 60 μL	24 μg/mL

Table 2: Details of the lentivirus packaging system.

Culture Dish	Seeding Density (cells/dish)	Medium (mL)	Plasmid System			Transfection reagent (µL)
Culture Dish	Seeding Density (cells/dish)	Medium (mL)	Target plasmid (μg)	PMD2.G (μg)	PSPAX2 (μg)	Transfection reagent (µL)
35 mm	6 ×105	1.5	1.5	0.5	1	6
60 mm	1.5 × 106	3.5	3	1	2	12
100 mm	4 × 106	8	8	3	6	34

Results

Figure 1: Production of concentrated virus and estimation of titer. (A) Fluorescence intensity of GFP (green fluorescence protein) after infecting HEK-293T cells with unconcentrated lentivirus (1x) and concentrated lentivirus (100x) (B) Comparison of the infection rates of unconcentrated lentivirus (1x) and concentrated lentivirus (100x) as detected by flow cytometry. (C) Titer estimation of concentrated lentivirus. Scale bars = 250 µm. Abbreviations: GFP = green fluorescent protein; TU = transducing units.

Materials

List of materials used in this article
Name	Company	Catalog Number	Comments
0.45 μM filter	Millipore	SLHVR33RB
95%/100% ethanol	LANBAO	210106	Stored at RT
Antibiotics(Pen/Strep)	Gibco	15140-122	Stored at -20 °C
Anti-TRP2/DCT Antibody	Abcam	ab74073	Host/Isotype: Rabbit IgGSpecies reactivity: Mouse/Human Dilution of 1:200 to use
DMEM (High glucose)	HyClone	SH30243.01	Stored at 4 °C
DMSO	Sigma	D2650	Stored at RT
FBS	Gibco	10270-106	Stored at -20 °CHeat-inactivated before use
Gelatin	Sigma	G9391	Stored at RT
GFP-PURO plasmids (Mitf, Sox10, Pax3, Sox2, Sox9 and Snai2)	Hanheng Biological Technology Co., Ltd.	pHBLPm003198 pHBLPm001143 pHBLPm002968 pHBLPm002981 pHBLPm004348 pHBLPm000325	Stored at -20 °C
Hematoxylin	Abcam	ab220365	Stored at RT
Lipofectamine 2000	Invitrogen	11668-019	Transfection reagent, stored at 4 °C
PBS (-)	Gibco	C10010500BT	Stored at RT
Phorbol 12-myristate 13-acetate (TPA)	Sigma	P8139	Stock concentration 1 mMFinal concentration 200 nM
Polybrene	Sigma	H9268	cationic polymeric transfection reagent; Stock concentration 8 μg/µLFinal concentration 4 ng/µL
Puromycin	Gibco	A11138-03	Stored at -20 °C
Recombinant human bFGF	Invitrogen	13256-029	Stock concentration 4 μg/mLFinal concentration 10 ng/mL
Recombinant human insulin	Sigma	I3536	Stock concentration 10 mg/mLFinal concentration 5 µg/mL