The author's describe their replicon and reporter as such:
We screened for host proteins involved in HCV replication using the Huh7/Rep-Feo subgenomic genotype 1b HCV replicon that we had previously adapted to high-throughput screening of small molecule libraries. This replicon is an RNA molecule that encodes the HCV nonstructural proteins (NS3 through NS5B) that are sufficient to mediate viral replication as well as a firefly luciferase-neomycin phosphotransferase fusion protein. Firefly luciferase activity is a linear function of replicon RNA copy number and therefore permits sensitive and rapid quantitation of HCV replication. We optimized siRNA transfection conditions using a luciferase siRNA.
Is this to allow for the luciferase-neomycin conjugate to leave the membranous webs common in picornavirus replication? I emailed the author and he responded with this:
Needed to stably transfect the luciferase as readout
Can someone help expand on this answer? I'm specifically wondering why the luciferase needed to be conjugated with neomycin rather than just stand-alone luciferase.
One thing that occurs to me is that they may have wanted to ensure that both luciferase and neomycin resistance were obligatorily linked, i.e., they wanted to avoid the situation that cells could have neo resistance OR luciferase rather than both. Not knowing the experimental setup, I am not sure if this is a consideration.
It is also possible that they simply had that vector already constructed in the lab for another purpose and simply used it in the current set of experiments.
David, you are right. I misread what Feo actually is: luciferase conjugated with aminoglycoside phosphotransferase as a method of inhibiting the antibiotic, in this case neomycin. For some reason I was thinking they had luciferase conjugated with neomycin itself which is why I was thoroughly confused.
The author responded my follow-up questions with:
Only used for selection of stably transfected cells.
Thank you for your help!
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