The overall goal of the following experiment is to generate recombinant influenza virus from plasmid DNA. This is achieved by transecting the eight influenza gene encoding DNA plasmids into a co-culture of 2 9 3 t and MDCK cells by using Lipectomy 2000 as a second step. The tissue culture supinate will be used to infect fresh MDCK cells and or 10 day old chicken embryonated eggs, which will amplify the virus present in the supinate from transfected cells.
Next we will harvest the tissue cultures supernatants and the Alan Toic fluid from infected chicken embryonated eggs. In order to confirm the presence of the virus by using an HA assay, results are obtained that show the presence of the virus based on the presence of agglutination. Hi, I'm Luis Martinez Rito in the Department of Microbiology and Immunology at University of Rochester.
Today I'm gonna show you how to rescue recombinant influenza viruses from plasmid DNA. We use this, this technique in our laboratory to study multiple aspects in the biology of influenza virus and to develop potential vectors and vaccines. So let's get us started.
Prior to starting transfect warm cell culture reagents to 37 degrees Celsius, one times phosphate buffered saline, P-B-S-E-D-T-A, trypsin and KO's modified eagles medium DMEM with 10%fetal bovine serum FBS and 1%penicillin streptomycin PS.Next, prepare 250 microliters of opti MEM media and six to eight microliters of LPF 2000 per transfection and incubate at room temperature for five to 10 minutes. Prepare the plasmid transfection mixture by adding one microgram of plasmid DNA to a tube with 50 microliters of opti MEM media. Add one microgram each of the eight PDZ plasmids containing viral segments.
PB two PB one pa ha np na m and N MS.Add 250 microliters of room temperature opti MEML PF 2000 into the influenza DNA plasmid transfection mixture and incubate for 20 to 30 minutes at room temperature. Meanwhile, prepare suspensions of 2 9 3 T and MDCK cells for transfection. The density of the 2 9 3 T and MDCK cells should be at 80 to 90%confluence.
The day of transfection, approximately five times 10 to the six cells per 100 millimeter dish Wash cells detach and spin down using standard cell culture techniques. RESUSPEND 2 9 3 T cells in three milliliters of D-M-E-M-F-B-S-P-S. When Resus suspended, deliver the three milliliters to the MDCK cells for resuspension.
This will create the mixture of 2 9 3 T and MDCK cells to be used as co-culture. Add 250 milliliters of the 2 9 3 T-M-D-C-K cells per well into a six well plate. After 20 to 30 minutes, add one milliliter of DMEM 10%FBS 1%PS to each of the opti M-E-M-L-P-F 2000 influenza DNA plasmid mixtures.
Then add into the wells with the 250 milliliters of 2 9 3 T-M-D-C-K cells. Gently shake the six well plate and let the transfection incubate overnight in the incubator at 37 degrees Celsius and 5%carbon dioxide. The next day, 16 to 24 hours post transfection, change the transfection media and incubate the transfected cells in DMEM 0.3%Bovine albumin or BA 1%PS containing one microgram per milliliter of L tosil lado two phenol ethyl chlor methyl ketone or TPCK treated trypsin for 48 hours.
If MDCK infection is desired plate fresh MDCK cells one day prior to infection or two days after the initial viral transfection, then 48 hours after changing the media transfer the SUP natant from the transfected cells into a micro centrifuge tube centrifuge. The tissue culture sup natant in a micro centrifuge for one to two minutes at 13, 000 RPMs prior to infection of the MDCK cells. Check the cells under the microscope to confirm a monolayer, then proceed with the infection Wash cells twice with one milliliters of one times PBS.
Add 200 microliters of centrifuge tissue culture supernatants from earlier steps and incubate for one hour at room temperature. Rock the plate every 10 minutes to avoid drying the cells after one hour of viral absorption. Remove the infection media from the MDCK cells and add two milliliters of DMEM 0.3%BA 1%PS containing one microgram per milliliter of TPCK trypsin.
A cytopathic effect will be observed in the MDCK infected cells at 48 to 72 hours after passage. Depending on the transfection efficiency and the virus load, cytopathic effects consist of cell rounding death and detachment from the surface, et cetera. Next, isolate snat from MDCK cells for the assay to confirm the presence of the virus in the tissue culture.
Supernatants micro centrifuge supernatants for one to two minutes at 13, 000 RPMs to remove cell debris and transfer to a new centrifuge tube to infect chicken embryonated eggs. Candle the 10 day old eggs using a light candling box to see the interface between the air sack and the Allen Toic cavity. Make a pencil mark on the interface border with a five milliliter syringe needle.
Make a hole in the egg shell with a one milliliter syringe. Infect each egg with 200 microliters of the tissue culture supernatants from earlier. Cover the hole in the eggshell with melted wax using a cotton swab.
Incubate the infected eggs at 37 degrees Celsius for two to three days prior to harvesting the Alan Toic fluid. Incubate the chicken eggs for two hours or overnight at four degrees Celsius to kill the chicken embryo and coagulate the blood following this. Wash the eggshells with 70%ethanol To establish sterile conditions.
Open the egg carefully over the air cavity by tapping with a spoon. Remove the broken eggshells using forceps with a one milliliter needle. Remove the Alan Toic membrane without breaking the eggs yolk.
Stabilize the chicken embryo with a spatula and guide a 10 milliliter pipet into the Alan Toic fluid. Collect as much Alan to fluid as possible. Eight to 12 milliliters into a 15 milliliter centrifuge tube on ice for each egg.
Do not break or collect any egg yolk centrifuge for five minutes at four degrees Celsius and transfer the Alan Toic fluid to fresh 15 milliliter centrifuge tubes on ice stall the tubes containing the centrifuge. Alan Toic fluid at four degrees Celsius until they are checked for the presence of rescued virus with an HA assay. The HA assay is performed using VBO 96 well plates negative controls using PBS one X and positive controls of tissue culture.
Supinate and or or Alan Toic. Fluid from a non-res influenza virus infection should always be included in NAHA assay. Dispense 50 microliters of PBS one X into each well of the V bottom 96 well plate add 50 microliters of the MDCK tissue culture supernatants and or Alan Toic fluid from the infected eggs to the first well and make twofold serial dilutions for the following wells.
Discard the extra 50 microliters from the last. Well add 50 microliters of 0.5 to 1%chicken red blood cells to each well incubate the V bottom 96 well plate for 30 to 45 minutes on ice or until a red. is visible in the bottom of a negative control PBS sample the presence of virus in the MDCK tissue culture supernatants and or in the LAN toic fluid from infected eggs can be determined macroscopically using hemagglutinin of chicken, red blood cells.
The presence of virus induces hem agglutination while the absence of virus allows the formation of a red pellet in the bottom of the well. Additionally, the existence of CPE cell rounding death detachment from the surface, et cetera in cells infected with the tissue culture supernatants or with the LAN toic fluid from eggs will suggest the positive viral rescue in the case of influenza virus, approximately 10 to the three to 10 to the four plaque forming units or pfu are required to give a positive signal in the HA assay. Therefore, an immunofluorescence assay can be performed in parallel with the HA assay to confirm a true negative result.
Because presence of individual viral particles can be detected by staining infected cells with fluorescently labeled antibodies, it is possible that HA negative s supernatants or Alan Toic fluids are positive when using immunofluorescence assays. In this case, the virus should be amplified by packaging again in MDCK cells or an eggs. Alan Toic fluid and or tissue culture supernatants from the second passage should now be clearly positive in the HA assay.
So we just saw you how to rescue rein and influenza viruses from plasmid DNA. The rescue of influenza viruses from plasma DNA is a simple and a straightforward process. One, the protocol is originally done in the laboratory for the rescue of recombinant influenza viruses from plasma DNA.
We recommend three independent transfect per recombinant virus. If more than one recombinant influenza virus rescue is attempted, scale the steps accordingly to the number of virus to be rescue. The following transfection and infection protocol to rescue recombinant influenza viruses from plasma DNA is a skill for six well plates.
All procedures to harvest LAN toic fluids from infected eggs are performed under sterile conditions. So that's it. Thanks for watching and good luck with your experiments.
