Production and use of lentiviral vectors based on feline immunodeficiency virus

Summary

Lentiviral vectors based on feline immunodeficiency virus (F IV ) are very useful for mediating integrated transgenesis into non-dividing human cells. This chapter focuses on describing methods for the production and use of the new generation of F I V vectors, discusses key features of F IV compared to other lentiviral vectors, and provides detailed protocols for large-scale/small-scale vector production. In addition, the practical application of recently identified species-specific retroviral constraints and the generation of non-integrated F I V vectors are also the subject of this chapter

Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".

Operation method

Production and use of lentiviral vectors based on feline immunodeficiency virus

Move

Production of vectors with cells grown in CFlO or CF2 devices Materials

reagents

Transfected cell line: 293T

Early stage 293T cells are better. They can divide consistently and uniformly before being grown for transfection.

DMEM plus 10 % fetal bovine serum (DMEM-10), lOO^/ml penicillin, lOOug/ml streptomycin, and 2 mmol/L L-glutamic acid were added to the cells.

Plasmid DNA: pMD.

For in vitro transfection, plasmid DNA should be sterile and endotoxin-free. Commercial kits such as the Plasmid Macro Kit for Endotoxin Removal (QIAGEN 2362) can be relied upon. Cesium chloride purification methods are also available.

Transfection reagents

Tris-HCl (10 mmol/L, pH 8.0) was used to dilute the plasmid DNA.

CaCl2 (2.5mol/L)

Autoclaved and stored at 20°C.

2X HEPES buffered saline (HBS)

Preservation solution-Na2HPO4: 52.5 g Na2HPO4, 5000 ml H2O0 2 X HBS: 80 g NaCK 65 g HEPES (sodium salt), IOOml Na2HPO4 preservation solution. Careful optimization of pH is very important. Adjust pH with l ml/L NaOH to a series of set points with narrow ranges, e.g. pH 6.95, 7, 7.05, etc. Adjust the final volume to 5000 ml. Adjust the final volume to 5000 ml. The optimal pH 6.95 is available. However, the final use of the storage solution is determined by experience in transfection efficiency. Freeze storage (1-20°C). The p H varies with time, so different transfection percentages can be obtained. Reformulation is required every 3 to 6 months. By transfecting plasmids expressing G FP, it was found that the optimal transfection reagent could transfect 95% of the 293T
cells. The transfection reagent can be refrigerated or frozen, but it is important to use the transfection reagent at room temperature during transfection.

Trypsin-EDTA (cell culture grade)

Equipment

Suction bottle (2L Kimax) (Kimble Glass 14607-2000) or similar suction equipment

Beaker (1L, same size as the volume of medium used)

Culture bottle (sterile plastic bottle; CF2: 250ml, CFlO: IOOOmD)

Cell factory with silicon tubing and connectors to culture bottles (sterile)

Funnel (sterile)

Incubator, preset to 37°C, 5% CO2 (racks should be precisely adjusted to fit the cell factory)

Microscope

Cells cultured in the CF2 plant can be viewed with most tissue culture microscopes. For the CFlO Cell Factory, a Nikon Eclipse TE300 or similar microscope is required.

Nunc Cell Factory (1-layer CF1, 2-layer CF2, 10-layer CFlO or 40-layer CF40)

Nimc Cell Factory Starter Kit (170769) with H D PE Connector (171838), Closure Lid (171897), Blue Seal Lid (167652) and Gelman 4210 Bacterial Air Vent Filter Membrane. Tissue Culture Flasks with Through-Air Lids (175cm2; T 175)

Methods

1. Preparation of cells and transfection

For C F lO devices

a. 3 days prior to transfection, inoculate 6 vials of T 175, each containing ^6x106 2Q 3T cells, and incubate for 48 hours.

b. One day before transfection, digest T 175 cells with trypsin and inoculate 2. ^5XIO8 293T cells into CFlO.

For C F2 device

a-Three days before transfection, inoculate 2~3 vials of T 175, each vial containing ¹⁰⁷ 293D cells, and incubate for 4811.

b- One day before transfection, digest T 175 cells with trypsin and inoculate 5X ¹⁰⁷ 293T cells into CF2.
2- Add 3 plasmids (pMD.G, pFP93, pGINSIN) in the ratio of 1:3:3.

For C F 10: Mix 84.5/253.5/253.5ug of plasmid into a 250ml sterile vial. Adjust the volume to 60. 5 ml with 100 mm0 1/L Tris-HCl ( p H 8. 0 ), add 6. 5 ml of 2. 5 m m o l / L CaCl2, and mix by swirling in the airtight bottle.

For C F 2 : Mix 16. 9/50. 7/50. 7 wow plasmids into a 2 50 m l sterile vial. Adjust the volume to 60.5 ml with 10 mmol/L Tris-HCl (p H 8.0), add 6.5 ml of 2.5 mol/L CaCl2, and then mix well by swirling in a closed bottle.

3-Tilt the apparatus to pour off the liquid inside the bottle, then add 67ml (CFlO) or yes 13. 4ml (CF2)2X HBS0

4. Immediately gently invert the bottle 4 to 6 times to homogenize (preferably by spinning). Precipitate the DNA for 3~5min.

5. Transfer the DNA-Ca2PO4 mixture to another container and stop the precipitation.
For CFlO: transfer to 800m l of fresh medium into -'a sterile plastic covered bottle of IOOOml. This bottle can be used with a filtration unit.

For CF2: transfer to 185m l of fresh medium into a sterile plastic 250m l covered bottle.

6- Replace the medium as follows:

a. Pour the cell factory medium into a dirty beaker and discard.

b. Attach sterile silicon tubing to the funnel and connect to the cell factory.

c. Set the cell factory aside so that the open vent is closed to the outer working layer.

d. Raise the funnel and slowly pour the transfection mixture and fresh medium into the funnel. Make sure it is evenly dispersed.
into each compartment.

e. Lift the cell plant at the end of the connection/vent tube so that the medium flows through the open vent tube opening, thus preventing the
leakage of medium from the upper compartment to the lower.

f. Arrange the cell plant accurately and horizontally on the incubator shelf (cells can be just covered by the transfection mixture).
covered by the transfection mixture).

7- Incubate the cells for 16 to 18 h and observe the outer layer of the tissue culture with a microscope. A better precipitation of D N A _hydroxyapatite
(a black spot much smaller than the cell) should be presented on the surface of the cell.

8- Replace the medium as follows:

a- Pour the cell factory medium into a dirty beaker and discard.

b. Attach sterile silicon tubing to the funnel and then to the cell factory, then set the cell factory aside.

c. Raise the funnel and slowly pour 1,000 ml (CFlO) or 200 ml (CF2 ) of fresh medium into the
funnel. Ensure that it is evenly dispersed into each compartment.

d. Lift the cell plant at the end of the connection/vent tube so that the medium flows through the open vent as in step 6.
opening as in step 6.

e. Place the cell plant accurately and horizontally on the incubator shelf.

Use cell harvesting and concentration carriers grown in CFlO or C F 2 equipment.

This protocol describes a method for harvesting and concentrating carriers from cells grown in a CFlO or CF2 device.

Materials

carve on trial basis

Bleach (10%)

Ethanol (70 % dissolved in tissue culture grade double distilled water, molecular grade)

Phosphate buffer solution (PBS, tissue culture grade)

Sucrose (20 %), 20 mmol/L T ris (pH 7. 4), 100 mmol/L NaCl solution
The solution was filtered through a 0.45um or 0.2pm membrane.

Equipment

Autoclave

Culture bottles (250 ml plastic bottles with screw caps) (e.g. Corning polystyrene culture bottles, 430281)

Cell filters (e.g. BD Falcon, 70um nylon, 352350)

Freezing tubes with screw caps (1.0 to 1.8 ml, sterile, Nunc)

Filter equipment (1000ml, 0.22um pore size) (e.g. Nalgene MF75 series)

Microcentrifuge tubes (I.5m l and 0.5ml, sterile)

Micro pipettes

Balanced centrifuge tubes with graduated scale

Ultracentrifuge (Sorvall Discovery 100SE, Beckman L8-80M, or equivalent)

Ultracentrifuge Bottle (250 ml): Heterogeneous Homocrystalline Polymer Ultracentrifuge Tubes, Oakridge (Sorvall 54477) with Fluorocarbon Cap (Sorvall 54421) and A612 Rotor (Sorvall 119977) or equivalent.

Ultracentrifuge Tubes (36 ml): Heterogeneous Homocrystalline Polymer Ultracentrifuge Tubes (Sorvall 03141) can be used in any capacity.

For use with SureSpin 630 (Sorvall 79367) or SW28 (Beckman 342207).

12 ml Homogeneous Heterocrystalline Polymer Ultracentrifuge Tubes (Sorvall 03699) for use with TH641 Rotor Head (Sorvall 08224) or SW41Ti Rotor Head (Beckman 341302).

Methods

1. 48h after changing the transfected cell culture medium, collect the supernatant in a large sterile container. Centrifuge at low speed for a few minutes to remove cell debris.

Other methods are available, such as sinking the isolated cells to the bottom of the bottle for 3~5min. The cell factory can be washed twice with autoclaved water in a tissue culture rack and then sealed and stored at 4°C for reuse.

2. Pour the supernatant into a 50jum cell filter to clarify the supernatant. The supernatant was then filtered through a 0.22um filter. An IOOOml Nalgene device is sufficient to contain 600~800m l of vector. A 500m l Nalgene device is sufficient for 300 ~ 400m l of vector before clogging. Dispense a small aliquot (~2 ml) of filtered carrier supernatant into a freezer tube for later titration and recovery control. Store to one 80°C .

The VSV-G pseudotyped FIV vector is very stable at higher temperatures for short periods of time, remaining half viable at 37°C for 24h or 4°C for 72h.

3 - Wash the appropriate rotor head to be used with 70 % ethanol.

4- Centrifuge the carrier supernatant as follows:
For CFlO
a. Wash the inside of a 250 m l homogeneous heterocrystalline polymer Oakridge ultracentrifuge vial with 70 % ethanol and the fluorocarbon cap. Blow dry.
Use reusable ultracentrifuge bottles and white caps for the A621 rotary head. These bottles should not be autoclaved; they should be sterilized with a 10% bleach solution.

b. Add 200 ml of filtered carrier supernatant to each 250 ml ultracentrifuge bottle. After filtration, the carrier supernatant
After filtration, the total volume of carrier supernatant will be less than 1L . Adjust the volume of the last bottle with PBS to 200 ml. close the lid tightly.
If these tubes or bottles are not full, they may partially collapse during ultracentrifugation. Balance these tubes with the caps using the balancing arm.

c. Centrifuge the tubes in a Sorvall Discovery 100SE ultracentrifuge (67,000g_ ) with an A612 head at 19,000r/min or equivalent at 4°C for 6h.

d- Pour off the supernatant and place the 250m l bottle at a 45° angle diagonally on ice with the carrier pellet facing down. Aspirate and
pre-cooled liquid in the opposite direction of the carrier pellet. Then, rotate the carrier pellet downward and add 6 to 7 ml PBS
to the set concentration for the second time. Otherwise, an appropriately small volume of PBS can be added.

e- Start the resuspension by adding P B S along that side of the carrier pellet with a pipette. First wash the full length of the tube directly with liquid
flow directly to the pellet. This step takes about 5 min for each bottle. add all the material to a 250 m l bottle to start the second ultracentrifugation.

Resuspend 35 to 4 Oml of carrier supernatant into 6 bottles. Some of the carrier from the first concentration can be aliquoted (e.g., 800ul) into tubes and frozen at 180°C for later titer and recovery comparisons. Retain 50 % to 80 % of the transduction units for the first concentration recovery.

f. For the second concentration, wash 12 ml of reusable homogeneous heterocrystalline polymer overdrive with nanopure water.
centrifuge tubes to remove paper fibers used for packaging that can be adsorbed and co-purified with the carrier particles. High Pressure Extinguishing
sterilized and cooled to room temperature. The tubes are not reusable. Ultracentrifugation thins the plastic and the tubes will leak if used a second time.

g- Wash the barrel and lid of the SW 41 T i rotary head with 7 0 % ethanol and blow dry. Add 9m l of resuspended carrier from the first concentration and Iml 2 0 % sucrose solution to 12m l of homogeneous heterocrystalline polymer ultracentrifuge tube. If there is
necessary add PB S to fill the maximum volume of the large disk of the tube (10 ml) and equilibrate tightly.

It is recommended that this step starts with the addition of 5m l of resuspended carrier to the ultracentrifuge tube, followed by the addition of Im l of sucrose solution directly to the bottom of the tube, and then the addition of 4m l of resuspended carrier to the top of the total volume. This technique ensures a suitable interface between the recovered carrier and the sucrose solution.

h- Disconnect also, I.5h in a Sorvall Discovery IOOSE ultracentrifuge with a SW41 T i rotor head or equivalent equipment at 24 000r/min at 4 C (the bottom of the bottle is 67 OOOgnnax, while the top of the bottle is 31 OOOgWi). the pellet will be visible again.

i. Resuspend and wash the bottom of the tube for 5 min with lOO^J P B S. The micropipette can be adjusted to a volume of 100 ul first. ii. Keep the tip of the gun below the liquid level to avoid air bubbles. ii. The resuspended liquid is milky and slightly viscous.
properties. Add the resuspended carrier to a 1.5 m l microcentrifuge tube and centrifuge at 3000 g for 3 min to remove the
The suspended material is not removed.

The second concentration recovery retains 30% to 70% of the transduction units compared to the original unconcentrated supernatant. Some recoveries can be as high as 1,000%, which reflects the degree of dispersion of the aggregated transfection units in the original supernatant.

j. Aliquot the vector pellet into a suitable volume (e.g., 50 jul or 100/^1) and freeze at 1°C.

k. Wash the A621 rotor head with 70% ethanol to remove any carrier supernatant that may have leaked out. Store at 4°C.

For C F2

a. Wash the inside of a new 36 ml - secondary polyallomer ultracentrifuge tube with nanopure water to eliminate the fibrous cardboard. It will not flush away any debris of any kind attached to the carrier particles. Autoclave the tubes and allow them to self
cool to room temperature before use.

Tubes are not reusable. Ultracentrifugation thins the plastic and the tube will leak if used a second time.

b. Add 25 m l of carrier supernatant to each 36 m l tube. Slowly aspirate 5 ml of 2 0 % sucrose solution and add it to the bottom of the carrier supernatant. Then add 2.5 to 3 m l of carrier supernatant to the maximum volume.

c. Centrifuge in a Sorvall Discovery 100SE ultracentrifuge (67 OOOgmax) using a SW 28 head at 19,000r/min or equivalent at 4°C for 1.5h. The pellet will not be visible at this point.

d- Aspirate the supernatant from the tube and retain a small volume. Resuspend with P B S or other pre-determined medium.
Add lOOul PB S or culture medium to each tube. Clean the walls of the tubes with a micropipette. Prior to resuspension use a gun
tip along the bottom of the tube. This step takes about 5 min for each tube. collect all the supernatant into one tube. The goal is to collect 1 to 2 m l of resuspended vector. Aliquot the vector particles into the appropriate volume (e.g., 1 to 2 ml).
50ul) and freeze at -80°C.

Retain 5 0 % to 8 0 % of the transduction units for the first concentration recovery.

e. Wash the drum with 70 % ethanol to remove any carrier supernatant that may have leaked out. Store the transducer head and bucket at 4°C.

Production and collection of carriers from cells grown in T75 cell culture flasks Materials

test splinter

Transfected cell line: 293T

Early stage 293T cells are better. They can divide consistently and uniformly before being grown for transfection.

DMEM plus 10% fetal bovine serum (DMEM-10), 100 U/ml penicillin, lOOug/ml streptomycin, and 2 mmol/L L-glutamic acid were added to the cells.

Plasmid DNA: pM D .G, pFP93, pGINSIN

Transfection reagents

Tris-H CK (10 mmol/L, pH 8.0) was used to dilute the plasmid DNA

CaCl2 (2.5mol/L)

Autoclaved and stored at 20°C

2X H EPES Buffered Brine (HBS)

See ingredients in Program 1.

Equipment

Filtration equipment (0.22um, 50ml; Nalgene)

Cell culture flasks with side-air access (75cm2, T 75)

Tubes (5 ml clean polystyrene tubes, e.g. Falcon 352058)

Vortex mixer for mixing transfection complexes in Falcon tubes

Methods

1... Two days prior to transfection, adjust the cell culture medium to be ready for transfection. The day before transfection, inoculate 3 X ¹⁰⁶ cells into each cell culture flask and grow overnight.

2. 4h before transfection, replace the growth medium with IOml of fresh medium.

3-Adjust the ratio of the 3 plasmid DNAs (pMD.G, pFP93, pGINSIN) to 1 : 3 : 3 , add l.O ug pMD.G, 3. Opg pFP93, 3. 0 wow pGINSIN to a sterile 5. Oml Falcon tube. The volume was adjusted to 800ul with 10mmol/L Tris-HCl (pH 8.0 ).

4- Add 800ul of 2.5mol/L CaCl2 while spinning at a moderate rate and then allow the CaCl2 to precipitate 3mol/L will appear a fuzzy milky white color.

5 . Aspirate the mixture and add it directly to the medium at the bottom of a tilted 1 7 5 culture flask so that the cell monolayer does not fall off. Shake gently to disperse the precipitate evenly into the cell monolayer without disturbing the cells.

6. Incubate for 16 to 18 h. Then, replace the medium with 10 to 15 ml of fresh medium by gently removing the medium by pipetting. Be careful not to disturb the cell monolayer.

7. Continue incubation for 48 h. Collect the supernatant by centrifugation and filter with 0-22um membrane.

8. Aliquot into freezing tubes and freeze at 80°C.

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