I have transfected a clone-derived virus that contain luciferase gene as a marker within the virus genome. I have tried to transfect the virus into human muscle cells but unfortunately i found that there is no significant CPE observed in the cells after few days post transfection.
again, i harvest the supernatant from the transfected cells and then re-infect into another flask of cells. same thing, the cells seems like overgrow until quite confluent, which again the CPE appearance becomes very ambiguous.
anyone has any idea or experience about this? Is it the virus clone is not stable with the marker gene or it somehow attenuates its replication becomes very slow. Any idea how to solve this cells overgrowth problem?
Just curious, did you succeed to infect the human muscle cells with your wildtype ones (without luciferase reporter gene)?
How did you determine where to insert the luciferase gene? As you wrote, it is possible that the reporter has completely disrupted an essential viral function. Or possible that there is replication but it is attenuated and no CPE occurs. Have you measured luciferase activity in your transfected muscle cells? Or can you measure some other product of viral infection (immunoblotting, immunofluorescence, viral genomes by qPCR/qRT-PCR)?
Christoph,
I am working on Enterovirus A71, which is one of the Picornavirus.
Andrew,
I has inserted the luciferase marker gene between the 5' utr and VP4 along with a cleavage site (as worked by some other researchers). i did measured the luciferase activity after transfection but there is no significance between mock and transfected cells.
Did you tried to transfect the cells without the luciferase insert? I mean the wildtype virus in clone format. And not the virus stock harvested by normal infectious.
How did you get the viral genome from (eg: PCR from culture extracted RNA, gene synthesis , etc) ? You propably selecting a non-viable EV-A71 strain for cloning. Did you check the sequences? Is the poly-A tail long enough?
I think you might just be unlucky to pick out the non-viable quasi-species strain of the virus.
My labmate has told me he did a few infectious clones (EV-A71) that are not viable. Sometimes a few amino acid mutation will make the virus to be defective. Please take note that quasi-species are common in Picornaviruses.
And finally, temperature sensitivity might be the factor (Eg: EV71-BrCr Ts and Tr strains).
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