In this paper, 293T cells were used to wrap virus in six orifice plates (35mm), and other orifice plates correspondingly increased or reduced volume.
Prepare for
Reagent paper
☑ core plasmid;
☑ index growth of 293 t cell;
☑ virus plasmid Mix packing: 1 (including g/(including l (Mix = pMDL: types - g: REV = 5:3:2), different carrier system used in the packaging virus plasmid is different also, this system can be used for packaging PBOBi, PLKO, Plv carrier such as plasmid.
☑ transfection reagents: turbofect, can also use other like lipo2000 etc.
1.Disc cells points
Collect cells by trypsin digestion, with the proper complete medium tile cells on the 35 mm petri dishes (according to the experimental need to choose a dish, after making the adherent cells by more than 80% of the total area of area reaches a dish).
Put cells at 37 ℃ temperature box containing 5% CO2 8-24 h incubation, when cells stick wall can begin after completely transfection.
Transfection of the previous 2 h fluid (1.5 ml full medium for replacing the old medium).
2.Mixed core plasmid and packaging plasmid
To remove 1.5 ml EP tube, add 400 l serum DMEM, add 1.5 g core plasmid and 1.5 g virus packaging plasmid, and 6 l turbofect (turbofect: plasmid = 2:1), fully mix well and rest 15-20 min.
3.incubation
Will chase the 400 mu l mixture drop join the monolayer cells in cell cultures, gently shake plate blending at 37 ℃ temperature box containing 5% CO2 after incubation.
4.Collect the virus and clear it
6-10 h after suction to medium, add 2.5 ml37 ℃ preheat complete medium, continue to place the cell incubation temperature box, approximately 40 h can collect including slow virus infection or repackaging the supernatant of the at - 80 ℃ storage.
(1500rpm centrifuge for 5 minutes, usually collected to 2ml containing virus)
5.Virus infection
1) the cells are spread out on six orifices or 12 orifice plates, and the optimum density is 30%-70%.
2) for six orifice plates: (12 holes halved)
The Infectmouse cells: 800-1600 l virus + 1600-800 mg l fresh medium = 2.4 ml total
Infecthuman cells: 200-800 l virus + 1200 mg l - 800 mg l fresh medium = 2 ml total
3) add 2-5 (including g/ml polybrene, fully shake after incubation at 37 ℃ temperature box.
4) change fluid after 12-24h, after being overgrown, overgrown, or detection expression, do the experiment.
In the experiment of virus packaging, researchers often encounter the virus packaging experiment with the same viral vector system, why some people do well and succeed the next time.
Some people do not come out from time to time, the stability is very bad, not the drop is low is not to be toxic at all?
Adenovirus or Lentivirus ?
Lentivirus can be inserted into the cell genome, stable expression, long duration.
The packaging cycle is short (usually two months), and the infection rate is much lower than that of the adenovirus.
No amplification. Once a packaged virus is used up, it can only be repackaged.
The number of viruses in a single package is also relatively low (10 to the eighth pfu/ml), which is not suitable for direct use in animal experiments.
Adenovirus infections are highly efficient, and many cells can reach nearly 99 percent.
The purified adenovirus can be injected directly into living animals.
It can be amplified in vitro. After each use, it can be used to expand the cell and save cost.
The number of drops in a package is high (10 to the 11th power pfu/ml, and 10 to the 10th power pfu/ml is not purified).
It's not integrated into the genome, it's not stable expression.
The expression time is shorter than the slow virus (two or three weeks).
The cycle of the packaging adenovirus can be longer than the slow virus (usually two and a half months), because the adenovirus packaging process is relatively cumbersome.
Key points of virus packaging
Virus several key nodes of the packaging are cell factors, carrier system (try to use mature commercial carrier system), construct recombinant plasmid is correct or not, plasmid extraction and purification, packaging transfection control (24, 48 hours of cells and a fluorescent state judgment), purpose gene effect on virus packaging (size, sequence, protein gene functions toxicity can will affect the packaging success).
How to improve the efficiency of virus infection
The virus infection efficiency is mainly related to three aspects, the degree of the virus itself, the cell type and the individual operation.
Here I exclude operational problems first, if you want to improve the efficiency of viral infection, can improve the virus first degrees, in the process of infection of the appropriate increase the amount of virus infection or by many times to join the virus infection.
Second, you can also increase the infection rate by adding a number of adjuvant reagents, such as polybrene.
In addition, you will need to add the appropriate amount of virus to different target cells so that you can improve the efficiency of the virus more accurately.
293T cells
The 293T cell is relatively fast, and it is easy to fall off from the wall in the generation, so it is not necessary to blow it, and to be on the safe side, we should try to play it as well as possible during digestion.
In addition, the cells in each dish are also spread evenly and should be replaced in time.
When the 293T cells packed slow virus, the transfection method preferred the cationic liposomes method.
The efficiency of the transfection of calcium chloride is relatively low and the toxicity is relatively low.
And the calcium chloride method is very strict with the pH value, almost no.
Cationic liposome method the effect is better, but the routine use of Lipo2000 cytotoxicity, rich can use low toxicity Fugene HD, less money can choose other company's Lipofiter cationic liposome product, the efficiency and Lipo2000 about, basic no toxicity.
Virus concentration
The virus can be collected twice, and can be collected at 48 h and 72 h.
If you don't want to trouble concentrating the virus, you can also not condense it, and directly use the collected virus as a medium for infecting cells, but it may not work very well.
In addition, the nutrition of the culture medium has already lost a lot of nutrients when it is collected, so the direct culture of the infected cells can damage the cell, so it is suggested that it should be concentrated and then infected.
How to improve the activity of adenovirus?
To improve the activity of adenovirus, the key should be in the process of virus packaging and amplification.
The purification process simply removes the defective virus and cell fragments, etc. which can cause the body's immune response. If the amplified virus is high, then the purification will be higher.
How does the virus survive after purification?
The purified virus is preserved with a DMEM without serum, and is usually stored at -80 degrees for a long time.
If you don't want to purify it immediately, you can put it at -80 degrees, and then purify it together.
Objective protein is expressed in the cell or in the cell, and the titer is performed according to the target protein expression.
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