And I have never been disappointed. At first, to eliminate cellular debris, 1 liter of ICF is divided into 6 bottles which is centrifuged for 10 minutes at 15, xg using a JA fixed-angle rotor.
This step can be omitted, but if done so, there will be more impurities during ultracentrifugation with the cushion method in the next step. The more the pretreatment, the easier and cleaner will be the subsequent operations. Moreover, if there is a large quantity of impurities, the virus gets enmeshed in them, which might decrease the yield. After removing cellular debris, the remaining supernatant is subjected to further centrifugation for hours at 30, xg using a JA fixed-angle rotor to get the pellets where viruses are.
If you are in hurry, you can use an ultracentrifuge instead, and process mL at a time with a Type 45 Ti fixed-angle rotor. You can rapidly centrifuge these samples either at 30, rpm for 2 hours or 40, rpm for 1.
Next, suspend the pellets in 26 mL of phosphate buffered saline PBS. Deciding in what volume of PBS the pellets are to be suspended requires experience and intuition. In general, never concentrate thicker than 1 to 10 at each step of purification. For instance, if your starting material is 1 L, you should not concentrate below mL; if you concentrate to say, 10 mL, the resulting product will be muddy.
For ease of handling you may want to opt for higher concentrations, but you will regret later. However, this is only a rule of thumb. The impurities can be further removed using sucrose cushion method with a large capacity SW 32 Ti swinging bucket rotor.
For this, dispense 4. When ultracentrifugation is finished under the conditions given in Fig. The white band can be collected by one of the three different ways; directly inserting a syringe into the band to extract the white band; collecting the white band after removing the unnecessary liquid layers just above it, or by separating the fractions by inserting a needle through the base of the tube.
In order to further increase the degree of purity, density gradient centrifugation can be performed using a small- capacity SW 55 Ti swinging bucket rotor. Density gradations are produced by layering solutions with different densities, and this procedure is often considered time-consuming. However, a density gradient can naturally be formed after ultracentrifugation even with only two layers. This is a little trick. Layer 0. Sixteen hours mean starting centrifugation in the evening of the previous day and collecting the next morning, but there is no need to be strict about the duration.
As you can see in Fig. The upper band comprises 1. The virus particles contain RNA. The blue band above is of the empty particles consisting only of proteins with no nucleic acids and because they are proteins, the buoyant density is around 1.
Since the high-density cesium chloride is used, it is important to wash the rotor thoroughly with lukewarm water after use to prevent pinhole-like corrosion. Mazzone HM. Download PDF. Contact Us. Sign in to view contract pricing Your shopping cart is empty. Qty Price View Cart. Added to cart View Cart. Osamu Nakagomi, Nagasaki University Graduate School of Biomedical Sciences Molecular Epidemiology In recent years, in virus research, it has become a standard practice to purify and analyze genomes and identify viruses from samples using commercial kits.
A kinetic study showed that, in sucrose gradients containing this buffer, the virus gradually migrated as a single peak of infectivity from a density of 1. The density change was shown to be reversible; after the removal of the Tris-EDTA buffer, rebanding of virus harvested at the heavy density resulted in its banding at the lower 1.
The data indicate that density change could not be explained on the basis of the loss of some component from the virus or on the basis of the failure of the virus to reach equilibrium. Thus, an efficient protocol for high-titer lentivirus purification that is easily achieved with a regular lab centrifuge is described. To measure the titer of the functional virus, the concentrated virus was added to the HEKT cells, and the percentage and relative intensity of the GFP-positive cells were measured with a flow cytometer Fig.
The fluorescent intensity of the transduced HEKT cells showed no significant differences in any of the concentrated viruses purified with various RCF tests. The concentration of viral genomes of the purified lentivirus was also measured using the RT-PCR method. To test whether the different transduction efficiency was due to the different centrifugation time, the different speed, or both, a set of experiments were performed to compare the concentration efficiencies among the lentiviruses purified at various RCFs for various durations.
The impurity of the lentivirus preparations is an important issue, especially in in vivo applications. Thus, silver staining experiments were performed to examine the lentivirus purity. As shown in Fig.
The viruses were enriched at a ratio and added to the HEKT cells at the volume indicated in the lower panels. The effect of the centrifugation time on the virus transduction efficiency was first examined. Next, whether or not the sucrose concentration in the centrifugation buffer could affect the virus enrichment was tested. The osmotic of the centrifugation buffer was between Without sucrose in the buffer, the transduction of the concentrated virus was very low.
Compared with other viruses, such as the Adeno-associated virus AAV , the lentivirus is more fragile and subject to damage during purification. The data showed that the virus concentrated in the slow mode was slightly but significantly more efficient Next, the effect of multiple freeze-thaw cycles on the infection efficiency of the virus was tested. The results showed that the infection efficiency after one round of freeze and thaw was reduced by The percentages black and mean intensities red of the transduced cells were plotted to reflect the effectiveness of the raw left or concentrated right lentivirus.
One of the major uses of the lentivirus is to infect non-dividing cells, such as neurons, for both in vitro and in vivo applications 14 , 15 , 16 , 17 , To test whether or not the concentrated virus may affect the growth of the neural cells, the miniature excitatory postsynaptic synaptic currents mEPSCs and miniature inhibitory postsynaptic synaptic currents mIPSCs of the infected neurons were measured and compared with the uninfected neurons.
In addition, the miniature synaptic transmission from the neurons infected with purified lentivirus at DIV 10 was measured when the synapses had already formed. The results show that the virus infection had no effect on the synaptic transmission, implying that the neurons in the late development stage DIV 10 were not affected by the virus infection Fig.
Using a stereotaxic injection, the concentrated virus was injected into the CA1 and CA3 regions of the hippocampus in newborn mice pups. The results show that no significant aggregation of GFAP-positive astrocytes were observed around the virus-injected sites in CA3 or CA1 region of hippocampus comparing to the uninjected hippocampus suppl.
Thus, these results showed that the virus enriched with our method is capable of expressing exogenous genes in the non-dividing cells. The purification of high-titer lentivirus is always a bottleneck for the application of lentivirus in a lab. Although other purification methods involving anion exchange HPLC 19 or ultrafiltration 20 have been reported, ultracentrifugation is still the most widely used method to concentrate lentivirus; however, the requirement of ultracentrifugation may represent an instrumental barrier in the concentration process.
Furthermore, this method can be easily modified for the large-scale preparation of lentiviral stock. The titer of the concentrated virus in the results varied between 2. Using the high-titer lentivirus 1. Figs 6 and 7. Moreover, the data showed that the concentrated virus purified using this method does not affect the synaptic transmission in cultured primary neurons, which is a sensitive cell preparation Fig. Several alternative methods have been reported to purify high-titer lentivirus preparations, which bypassed ultracentrifugation 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , Ion exchange chromatography was used to purify lentiviral particles based on the fact that lentivirus are retained by the anionic exchange columns and can be eluted subsequently with buffers containing a high concentration of salts 19 , 25 , This method has the advantage of removing more cell debris compared with the ultracentrifugation.
Polyethylene glycol PEG precipitation is another simple and efficient way to purify large volumes of lentivirus 21 , The method presented in this study is relatively simple and does not require special instrumentation or handling. In summary, a relatively simple and practical method to purify a high-titer and high-quality lentivirus for both in vitro and in vivo applications has been described. The dissociated hippocampal neurons were prepared from P0 pups, as described previously The cells were washed once using PBS and digested with 0.
The data were analyzed using FlowJo 7. Raw and concentrated virus proteins were extracted in the cell lysis buffer. Then, the gel was washed with distilled water three times. After three washes with distilled water, the developing solution was added to the gel for six minutes, and the stop solution was added to terminate the reaction. In brief, The total RNA The copy number of the lentivirus was calculated based on the standard curve. Electrophysiological recordings were performed in whole-cell mode, and the synaptic currents were monitored with a Multiclamp A amplifier Molecular Devices.
Patch pipettes were pulled from borosilicate glass capillary tubes World Precision Instruments using a P pipette puller Sutter Instrument. New born CD-1 pups P0 were used in this study.
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