The overall goal of this procedure is to propagate and detect an infectious clone of Mildy vs. No virus that expresses green fluorescent protein. This green fluorescent protein has been cloned into the gene coating for D utp Ace in the Myy Ner virus genome.
The infectious clone is contained in two plasmids. First, the two plasmids carrying the myy Ner sequences are cut with xpa one restriction enzyme and ligated. Second sheep choroid plexus cells are transfected with the construct.
Finally, myy vino virus infected cells are detected by fluorescent microscopy and flow cytometry. Hi, I'm Stefano Neron at the TER Institute for Experimental Pathology at the University of Iceland. Today we will show you a procedure for propagating and detecting an infectious molecular clone of my vascular virus that expresses green fluorescent protein.
The molecular clone is contained into plasmids P eight XSP five EGFP, and P six seven or of 12 kilobases and 4.5 kilobases respectively. Cut equimolar quantities of the two plasmids, namely 4.4 micrograms of P eight XSP five EGFP, and 1.6 micrograms of P six seven or with XPA one and ligate before transfection. When ligation is completed, incubate the ligation mix at 65 degrees Celsius for 15 minutes two days before transfection seed 10 to the six SCP cells in a T 25 tissue culture flask and grow cells for two days in five milliliter DMEM plus 10%lamb serum and two millimolar glutamine without antibiotics to a monolayer of 90%co fluency.
If the cells have not reached 90%CO after two days, leave the cells to grow one additional day. The lamb serum can be substituted by fetal bovine serum FBS. We routinely use lamb serum since some strains of MVV are inhibited by FBS.
This MVV strain, however, is not inhibited by FBS. When the cells are ready for transfection change medium to DMEM with 1%serum lamb serum or FBS without antibiotics. We use Lipofectamine 2000 for transfection, dilute the DNA in 500 microliters of opti MEM medium dilute 20 microliters of lipofectamine 2000 in 500 microliters of opti MEM medium, and leave at room temperature for five minutes.
After the five minute incubation, combine the diluted DNA with diluted lipofectamine 2000 total volume 1000 microliters. Mix gently and incubate for 2230 minutes. At room temperature, add the transfection mix to the T 25 flask containing SCP cells and medium mix by rocking gently.
Incubator 37 degrees Celsius and 5%carbon dioxide overnight. Change medium the following day to DMEM with 1%serum two millimolar glutamine, 100 IU per milliliter, penicillin, and 100 IU per milliliter.Streptomycin. Monitor the production of GFP and the SCP cells by inverted fluorescent microscopy.
Low transfection efficiency can be expected with these primary cells, usually five to 15%Incubate further at 37 degrees Celsius, 5%carbon dioxide for several days to allow spreading of the virus in the flask. It usually takes seven to 14 days for full infection. Harvest the virus by Ali, watting the supernatant into einor tubes.
Spin in a micro fige at 3000 or PM for five minutes. To remove cell debris. Transfer the supernatant containing the virus to new tubes.
Store at minus 80 degrees Celsius seed three times 10 to the four SCP cells. In 100 microliters of DMEM medium supplemented with 10%serum two millimolar glutamine, 100 IU per milliliter penicillin, and 100 IU per milliliter. Streptomycin per well.
In a 96 well plate incubate at 37 degrees Celsius, 5%carbon dioxide. If the cells are confluent the following day, change the medium to DMEM with 1%serum. Having 100 microliters in each, well dilute the virus in the following way.
Add 180 microliters DMEM with 1%serum per well to four times 10 wells in a 96 well round bottom or V bottom plate for serial tenfold dilution in quad duplicate. Add 20 microliters of virus to each of the four wells in the first column of the plate. And using a four channel pipee pipet up and down to ensure the samples are thoroughly mixed.
Change pipette tips and transfer 20 microliters to the next four wells and so on. Leave the last wells without virus for a negative control. Add 100 microliters of the virus dilution to the SEP cells and medium incubator.
37 degrees Celsius and 5%carbon dioxide. Observe for GFP fluorescence and cytopathic effects for two weeks. Cytopathic effects consist of cells becoming rounded and less transparent than normal cells with processes stretching out, ending with multi nuclear cells appearing and cell death.
Calculate virus titer using the Reid Munch method for rapid monitoring of replication of the virus. Infected cells can be examined by flow cytometry trypsin. Eyes around 10 to the five infected cells.
Wash once with PBS and RESUSPEND in 500 microliters. PBS add 500 microliters of 4%Para formaldehyde solution 2%final concentration incubated room temperature for 30 minutes. Pellet cells at 3000 or PMM for five minutes.
Wash with PBS two times for five minutes and analyze on a fact scan analyzer. Count 10, 000 events. The GFP expressing molecular clone of my virus presented here can be useful for analyzing virus host interactions and pathogenicity of my neurovirus, both in vivo and in vitro.
Although it has not been shown that my neurovirus can in affect human cells, it is important to observe all biosafety level precautions both cells and plasmid can be obtained from our laboratory. Good luck with your experiments.
