Published online Sep 8. Author information Article notes Copyright and License information Disclaimer. Received Mar 31; Accepted Aug This work is licensed under a Creative Commons Attribution 4. This article has been cited by other articles in PMC. Abstract Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Open in a separate window. Figure 1. The examination of purification parameters on the concentration efficiency of the lentivirus.
Figure 2. Figure 3. Figure 4. Discussion The purification of high-titer lentivirus is always a bottleneck for the application of lentivirus in a lab. Flow cytometric analysis The cells were washed once using PBS and digested with 0. Silver staining Raw and concentrated virus proteins were extracted in the cell lysis buffer. Electrophysiological Recordings Electrophysiological recordings were performed in whole-cell mode, and the synaptic currents were monitored with a Multiclamp A amplifier Molecular Devices.
Additional Information How to cite this article : Jiang, W. Supplementary Material Supplementary Information: Click here to view. Acknowledgments Supported by grants for Drs. Footnotes Author Contributions W. References Tang H. Lentivirus replication and regulation. Annu Rev Genet 33 , — In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science , — A third-generation lentivirus vector with a conditional packaging system.
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The resuspended pellet will be viscous if this step is done correctly see Note 8. Optionally, add one-tenth the volume of each pellet of Tracking dye. This can be left for 10 min with the resuspended PEG pellets, and will penetrate virions to fluorescently label the RNA inside without any additional permeabilization. This will make it possible to locate the virus-containing fraction or section of the pellet in any subsequent step, simply by resting the tube in a bottomless tube rack or clear beaker and illuminating the sample briefly with a UV transilluminator.
This step is useful for troubleshooting the purification procedure. It is important not to disturb the gradient layers at this stage. In this manner, overlay the resuspended PEG pellet carefully onto the sucrose gradients.
Since the pellet will be compact, it is not important to brake the centrifuge slowly. After centrifugation, decant and discard supernatants immediately. Invert the empty tubes on an absorbent surface for 5 min, and tap gently to wick away any remaining sucrose solution that may have gathered near the rim of the centrifuge tube. Do not use a pipette to resuspend the virus, as this may shear spikes and damage fragile viral envelopes see Note 9. Use a P pipette tip from which the pointed end has been cut off to gently transfer the virus suspension to a cryovial with minimal shearing.
Discard any insoluble material that remains, as most of the authentic virus will resuspend quickly and easily in comparison. At this stage, the virus should be monodisperse, and can be formalin inactivated if desired. Purified virus for mass spectrometry should not be formalin fixed, but can instead be inactivated using the solvent that will be used for mass spectrometry, provided this has been validated for your virus see Note This alternative method is suitable for purification of viruses that grow to lower titers.
Serum-free culture and preparation can also be used to remedy solutions that fail for cryo-EM or proteolysis due to high viscosity, non-viral protein contamination, or large amounts of insoluble material. Percentage recovery will typically be lower than that with Subheading 3. Perform steps 2—15 of Subheading 3. The PEG-protein pellets should be white, and may be quite small and susceptible to resuspending quickly upon standing for even a few minutes. Decant and discard the supernatants immediately.
Perform steps 16—22 of Subheading 3. The final translucent pellet may be small and quite difficult to see, but the presence of the virus can be confirmed using the Tracking dye and a transilluminator after removing the supernatant, if the dye was added at step 16 of Subheading 3. The number of infectious virus particles in the final preparation should be directly assessed by plaque assay or similar means as a retrospective measure of quality. The quickest way to assess sample quality is by EM of a negatively stained sample.
Ideal samples for proteomics or cryo-electron microscopy should contain a high density of virions with intact spikes, and relatively little non-viral material.
This protocol describes how to prepare a negatively stained coronavirus specimen for transmission-EM. Lay down a piece of paper towel on a work surface that is designated for work with slightly radioactive materials. Label the paper towel with the names of your samples.
Lay a piece of Parafilm M large enough to cover your labels, backing side facing up, on top of the paper towel and separate the backing from the Parafilm M at one corner. Trace lines through the backing paper on a piece of Parafilm M using the back of a pair of EM forceps to create channels for droplets of stain or wash buffer. The places where lines intersect will naturally hold droplets, and the labels will help keep the samples organized.
Peel off the rest of the backing paper, leaving the Parafilm stuck to the paper towel. Highly viscous samples will give a cleaner appearance after one or more wash steps, but washing is not necessary if the sample is quite pure see Note Do not reuse buffer or stain droplets, to avoid sample contamination. In between droplets, dry the grids.
Hold the grid perpendicular to a clean piece of filter paper and touch the edge of the grid to the paper. Most of the wash buffer should be carried off. After the washing steps, float each grid on a uranyl acetate stain droplet for 1 min. Longer staining will lead to more particles that appear to fill up with stain, if this is desired. Touch the edge of the grid to the filter paper several times to remove excess stain s ee N ote Samples should be visualized immediately, and can be best stored for longer periods in a grid box that is kept under a vacuum.
Ideal preparations for both proteomics and cryo-electron microscopy will contain monodisperse virions as opposed to clumps of virions, few smooth-walled exosomal vesicles, little or no stringy released ribonucleoprotein, and will have virions covering roughly one-tenth of the grid surface area. If possible, western blot analysis should be performed against the structural proteins of the virus to confirm the presence of viral proteins in the final preparation.
If infectivity of the virus is important, the number of infectious virus particles in the final preparation should be directly assessed by plaque assay or similar means as a retrospective measure of quality. As recent proteomic studies have shown, the presence of host cell proteins associated with coronavirus virions is to be expected, but a prominent band between 40 and 50 kDa is indicative of N protein. Protease digestion of purified virions can be used to remove proteins outside the coronavirus virion.
This should be optimized for each virus and each protease individually. It should be noted that after digestion, removal of extra-virion proteins would lighten the density of the virion and therefore a longer centrifugation is required to repurify virus particles. A wide variety of methods are available to study the coronavirus proteome. Gel based methods are still common and virions purified by these methods are compatible with gel based analysis.
Direct LC-MS analysis of coronavirus virions is also possible. Larger molecular weight polyethylene glycol preparations tend to be too heterogeneous to give the best results. One-well plates available from Nalgene can a cost- and space-effective alternative to standard tissue culture flasks for large-scale virus preparation.
Due to the simplicity of the proteome of the allantoic fluid of embryonated chicken eggs and the high titer of IBV that can be recovered, lower volumes of starting material can be expected. Early time points containing little virus can be discarded. Both sample purity and virus recovery are dependent on concentration. Virus can be collected at 2 h intervals beginning 48 h after inoculation to increase the final yield.
However, for best results, each virus sample should be processed immediately. In general, IBV inoculated into embryonated chickens eggs can be harvested from 16 h after inoculation, but can only be harvested at a single time point. The use of lower-concentration sucrose cushions can be used to study wild-type virions and virus particles containing defective mini-genomes that are of lower buoyant density [ 5 ].
Decanting quickly reduces the likelihood that some components of the pellet will resuspend. Ideally pellets should be separated from supernatants within 1 min of the end of centrifugation. In general, rapid resuspension, cold temperature, and the minimization of mechanical stress will all improve the quality of the preparation.
Soluble proteins contained in the pellet may alter the color to a yellowish hue. The translucent virion pellet may be difficult to see and will probably not be visible before the supernatant is removed.
The presence of the pellet can be confirmed by fluorescence under UV light after the supernatant is decanted, if the tracking dye was used. If a pellet is present, the viscosity of the added HEPES-saline will increase noticeably upon resuspension. Inactivation techniques should be validated beforehand. Both formaldehyde and 2-propiolactone can lose effectiveness over time. Amines in some buffers such as Tris—HCl will react with formaldehyde, which is why HEPES buffer is recommended for use throughout the purification process.
If left in contact with the supernatant, serum-free PEG pellets will resuspended much more quickly than serum-containing pellets, and the sample may be lost. It is therefore very important to decant the supernatants quickly with serum-free pellets. Alternatively, the sample can be applied as a droplet on which the grid is floated for 5—10 min.
Grids that continually sink in the saline or stain droplets likely have suffered extensive damage to the support surface and should not be used. It is therefore important to remove all excess uranyl acetate before visualization. If there is excess stain present, you will notice because the grid will stick to the forceps. If this happens, give the grid one more blot by gently touching the face of the grid containing the sample directly onto the filter paper, dry the forceps with another piece of filter paper, and try again.
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