hi, I am construction the Dengue Replicon cells, i found something very strange and i can not explain it, could you please help me?
The puromycin and Renilla luciferase gene are construct into the positive strand RNA and transfected into the cells, the puromycin resistance cell were collected and tested. Strangely, some of them do not have the Rluc activity.
I have transfected the RNA into 3 kinds of cells, BHK21, Vero and Huh7, almost each of the monoclonal BHK21 cells have the activity; only some of the monoclonal Vero cells have the activity, for those Vero cells do not have the activity, i keeped culturing them and then they got the activity; non of the Huh7 cells have the activity.
all the cells survives and grows good in the media contain puromycin (5 ug/ml ), and all the control cells died out in 3 days.
I want to get the Huh7 Replicon cells and i have keeped culturing them in the puromycin media for months, but they still do not have the activity.
Thank you for your answer, could you please tell me more detail? Do your explanation applies only to the situation that i want to establish the replicon in Huh7 cells? The early HCV replicon established were use the neomycin gene.
Since i have already established the BHK21 and Vero cell line, and there are other papers proved the feasibility of puromycin replicon system.
http://www.ncbi.nlm.nih.gov/pubmed/?term=Construction+and+characterization+of+a+stable+subgenomic+dengue+virus+type+2+replicon+system+for+antiviral+compound+and+siRNA+testing
i doubt if it is the Renilla gene is too toxic for the Huh7 cells and and want to replace it with EGFP to confirm the expression, do this helpful?
i would also try to replace Renilla with neomycin.
Hi Rui, Have you tried to detect viral proteins in HuH-7 cells by western blot or IF? I am wondering if your cells still harbor replicons, but somehow RLuc gene is non-functional. Or alternatively, the replication levels might be too low to allow detectable luciferase signals. Remember, puromycin acetyl-transferase is very potent in that even small amounts of this protein are protective against puromycin, but you have to have pretty good replication before the renilla luciferase activity becomes detectable.
It is interesting that some vero cell clones acquired the detectable luciferase activity after multiple passages. This could be due to the accumulation of cell culture-adaptive mutations that increased replication of replicons in those cells. Did you get the chance to sequence these replicons?
Hi, Mohsan, i have done other experiments before, I perform q-pcr to compare the replicon RNA copy numbers of the "activity" and the "non-activity" vero cells, it showed that the former being significantly higher than the latter, 10k vs 200 per cell. Our lab do not have the factors to perform WB or IF. I have reverse transcript the RNA and ready to send for NGS right now hope to get adaptive mutation site.
your answer and my experiment result indicate that the Huh7 cells seems do not have enough copies or adaptive mutations. i have picked more than 30 monoclonal cells but are still like this. i also use the total RNA from "activity" Vero or Bhk21 cells to transfect back to Huh7 cells, i think these RNA are better since they all ready have the adaptive mutations, but the result unchanged.
And Andrei, we may not try the firefly, as my boss told me it is more toxic than renilla, from his experience constructing the HCV replicon when he was in Rice's lab.
Hi Rui, who is your boss? I am also from Rice Lab.
Have you ever tried infecting your HuH-7 cells with DENV? There are a large number of HuH-7 cell clones circulating in various labs, and I wonder if you have, by chance, picked a wrong clone that is not permissive to DENV. I like your experiment where you transfected RNA from the BHK21 cell clones into HuH-7 cells, and the fact that this experiment did not work makes me believe that your cells might be screwed up.
My supervisor is Alexander Kolykhalov, he was one of the first researchers who characterized the genomes of HCV.
We do have considered the cells maybe the problem, we are trying to get new cells from other labs and the companies. And just 2 days ago i also electroplated the HpG2 cells, hope can get some results.
Thanks Rui for letting me know. Lets hope that new batch of cells would solve the problem. Please keep us posted.
Andrei, we have generated many HuH-7 cell lines expressing RLuc at various levels, and don't really see any toxic effects. Therefore, I would be really surprised if Rui's problem is due to RLuc. I like your idea of using NPTII gene (G418 selection) though. I always think that slow action of G418 provides comparatively longer time for replicons to accumulate adaptive mutations and acquire resistance to drug. However, it is interesting that puromycin selection worked fine in BHK21 and Vero cells, but not in HuH-7 cells. This could be due to differences in the replication kinetics in HuH-7 cells, or alternatively as I suggested in my previous post, the cells could be screwed up.
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