Cesium Chloride-Based Purification of Genome-Containing Adenoviral Vectors

Abstract

Source: Ehrke-Schulz, E., et al. Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5. J. Vis. Exp. (2016)

This video demonstrates the purification of high-capacity adenoviral vectors using cesium chloride gradient ultracentrifugation, enabling the separation of genome-containing virions for applications in gene delivery, vector characterization, and preclinical therapeutic studies

Protocol

1. Purification and Dialysis of HCAdV (High-capacity adenoviral vectors)
   1. To prepare viral lysate for cesium chloride (CsCl) gradients, freeze the cell-virus suspension in liquid nitrogen and thaw in a water bath at 37 °C 4x.
   2. Centrifuge the viral lysate at 500 x g for 8 min at RT (room temperature) and collect the supernatant containing the HCAdV (high-capacity adenoviral vectors) .
   3. For ultracentrifugation, prepare CsCl solutions as follows. Weigh 37.5 g (for 1.5 g/cm³), 33.5 g (for 1.35 g/cm³), and 31.25 g (for 1.25 g/cm³) of CsCl powder, respectively, and fill up with dH₂O to 25 ml.
      1. Stir until the solution becomes clear and sterile filter the solutions. Finally remove 1 ml of each solution and weigh it on a fine scale to check whether the density is correct. 1 ml should weigh 1.5 g, 1.35, and 1.25 g respectively.
      2. If the density is too high, adjust it by stepwise addition of small volumes (µl) of sterile dH₂O. After addition of dH₂O measure the density again. If necessary add more dH₂O.
      3. Repeat the procedure until the density is correct. If the density is too low, adjust it by adding a small amount of CsCl powder. Sterile filter it again and check the density. If the density is too high, adjust it by adding dH₂O as described before. If the density is still too low add more CsCl, sterile filter it again and check the density. Repeat the procedure until the density is correct.
   4. Prepare CsCl step gradients in 6 clear ultracentrifuge tubes. Carefully and slowly pipette CsCl solutions into the tubes using the following order: 0.5 ml of 1.5 g/cm³ CsCl solution, 3 ml of 1.35 g/cm³ CsCl solution, and 3.5 ml of 1.25 g/cm³ CsCl solution (Figure 1C).
   5. Overlay ~4.5 ml of cleared vector supernatant from step 1.2 on top of the 1.25 g/cm³ CsCl layer. Centrifuge the gradients in an ultracentrifuge using a swing-out rotor (SW-41) at 12 °C for at least 2 hr at 226,000 x g (35,000 rpm) with slow acceleration and deceleration to separate HCAdV-genome containing viral particles from empty particles and cell debris.
      NOTE: Under optimal conditions a diffuse band of cell debris formed on top of the tubes. Below, two white bands can be observed. The upper band contains empty particles whereas the lower band equals the HCAdV (Figures 1C and 2A).
   6. Carefully remove the layers of cell debris and empty particles and collect 1 ml of the lower bands from each tube and transfer virus with a clean pipette tip into a sterile 50 ml tube. Add up to 24 ml of 1.35 g/cm³ CsCl solution to the collected virus particles and mix carefully.
   7. Fill centrifuge tubes with 1.35 g/cm³ CsCl-virus solution to the top and centrifuge O/N (overnight) (18-20 h) at 226,000 x g (35,000 rpm) at 12 °C in an ultracentrifuge using a swing-out rotor (SW-41) with slow acceleration and deceleration.
   8. Collect the HCAdV present in the prominent lower band. As a potential upper band contains empty particles, remove upper layers from the top using a pipette (see Figures 1C and 2B). Then, take away the lower band using a pipette.
   9. Dialyze the collected virus particles for buffer exchange.
      1. Cut off a strip of dialysis tubing (MWCO: 50,000) of about 8 cm in length, wash it with sterile dH₂O for 3x. Then close one side of the dialysis tubing with a plastic clamp and transfer the collected virus from step 5.8 into tubing using a 1-ml pipette. Avoid air bubbles within the tubing and close it on the other side with a plastic clamp dialysis closure.
      2. Dialyze in 1-L of dialysis buffer (10 mM Tris-HCl (pH 7.5), 10% glycerol, and 1 mM MgCl₂ (magnesium chloride) in deionized H₂O) for 2 hr at 4 °C with slow stirring. Exchange dialysis buffer with 2 L of dialysis buffer and dialyze O/N at 4 °C with slow stirring. Alternatively, use a sucrose buffer (140 mM NaCl (sodium chloride), 5 mM Na₂HPO₄.2H₂O (di-sodium hydrogen phosphate, 1.5 mM KH₂PO₄ (potassium dihydrogen phosphate), and 730 mM sucrose, pH 7.8).
      3. Collect dialyzed virus particles from step 1.9.2. using a 1 ml pipette. Prepare multiple aliquots of desired small volumes (25-100 µl) and store purified virus at -80 °C.

Results

Figure 1. Representative results for the cloning procedure and release of the HCAdV (High-capacity adenoviral vectors) genome from pAdVFTC. (A) Release of gene of interest (GOI)-expression cassette from pHM5 by PI-SceI and I-CeuI digest. (B) purified GOI-expression cassette and purified, dephosphorylated pAdVFTC digested with PI-SceI and I-CeuI, respectively. (C) Analytical HincII digest of pAdVFTC clones with and without GOI. (D) Release of the HCAdV-genome carrying the GOI from pAdVFTC-GOI by NotI digest

Figure 2. Representative results after cesium chloride (CsCl) gradient centrifugation. (A) After performing a step gradient (see also step 1.5); (B) After continuous gradient (see also step 1.7).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ultra Clear Ultracentrifuge tubes	Beckmann Coulter	344059	density gradient centrifugation
Ultracentrifuge	Beckmann Coulter		density gradient centrifugation
SW-41 rotor	Beckmann Coulter		density gradient centrifugation
Spectrum Laboratories Spectrapor Membrane	VWR	132129	dialysis tubing
Ready-to-use dialysis cassettes	Thermo	66383	dialysis
Cesium chloride	Carl Roth	8627.1	density gradient centrifugation
LB medium	Carl Roth	X968.3	bacterial growth medium
Tris-HCl 99%	Carl Roth	9090.3	dialysis buffer/ lysis buffer
Glycerol 99.5%	Carl Roth	3783.1	dialysis buffer
MgCl₂ 98.5%	Carl Roth	KK36.2	dialysis buffer
NaCl	Carl Roth	3957.1	optional dialysis buffer
KH₂PO₄	Carl Roth	3904.2	optional dialysis buffer
Sucrose	Carl Roth	9286.1	optional dialysis buffer
Na₂HPO₄·2H₂O	Carl Roth	4984.2	optional dialysis buffer
1.5 ml tubes	sarstedt	72,730,005	storage of virus preparations at -80 °C