How to test if a cDNA clone from a plasmid is infectious? - (Apr/10/2008 )
Hi! Would like to get some input.
How would you put up your experiments if you want to test if your clone of a virus which you now keep in a plasmid is infectious?
How would you put up your experiments if you want to test if your clone of a virus which you now keep in a plasmid is infectious?
Hi Ammie,
Not trying to be rude, but is this a university lab project/homework question? As there's a different section for those queries and, if so, it helps to suggest some ideas so people feel that not giving you an unfair advantage and hindering your education. The answer to your question lies in what form of nucleic acid the virus you're studying uses in its genome.
Best wishes,
Ceri
hi! No, this is not any homework question, but had some kind of idea that I would get more answers if I did not specify it more... I just wanted to hear how different people solve this, and it would be interesting whatever virus.
But in my case I have a positiv-sense RNA virus.
There is no good known cell culture system for it so it is difficult to grow in cells, but some researchers have detected replication or viral proteins in some cells. So even if it can not be proved to get infectious it is interresting if replicating.
I have transfected cells through electroporation with capped RNA transcript of the viral sequence from the plasmid.
I have tried
1) just an ordinary RT real time PCR, to see if the amount if RNA gets higher with time. (It did not, so either there is no replication or it is hidden in the large amount of transcript used for transfection that is still there).
2) made cDNA using one of the primers, which theoretically should give sense-specific cDNA. (the antisense RNA is indicating replication) But it seems like the PCR is not sense-specific at all.
3) (or have actually not really done it yet, will do it tomorrow), immunofluorescense using serum/antibodies against the virus.
So if it do not work it could be that 1) the clone is not infectious 2) the transfection didn't work 3) the detection is not sensitive enough... so it feels that it could be done in a better way...
So back to the question: what would you have done. Controls?
Ceri
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This is really interesting. You are saying that there is a right answer to my question. Please tell me!
Noone that could give me some input into this?