What prevents a single-round lentivirus from transducing the cell line in which it was generated?
I am using GP2-293 cells as a packaging cell line to create lentivirus with the VSV-G surface protein. I will use the lentivirus to transduce hepatocytes.
In general, how does a lentivirus enter say, hepatocytes? The VSV-G surface protein binds to an LDL receptor. However, I thought that LDL receptors are only present on human bronchial epithelial cells and adrenal gland and cortex tissue. Therefore, how does a lentivirus with VSV-G enter hepatocytes?
If the produced lentivirus can also transduce the packaging cell line, it would not be optimal, as viral titer would be decreased after a certain time post transfection with viral components.
Additionally, if there is a broad spectrum of cells which can be transduced, is there a possibility of transducing one's own cells while working with such a virus?
Yes it can. There's a bit of attrition in your titer due to this reinfection process. In our lab we try to minimize this by harvesting the supernatant more frequently, we do 4 collections starting from 48h post-transfection (ideally every 12 hours but more realistically it's once in the morning when you arrive and once in the evening before you leave for the day).
As for LDL receptor, you're right that it's most prominently expressed on adrenal and bronchial epithelial tissue but it's still expressed at a low level across many tissues. My guess is that the VSV-G protein is just so high-affinity for the LDLR that not very much needs to be present on the target cell surface for the virus to actually enter cells.
Finally, yes, you definitely can infect your own cells with lentiviral particles. That's why you've gotta be careful! It's unlikely though, you'd need to allow virus a few hours of exposure time to your cells without washing it off to result in productive infection. Also, current engineered lentivectors are replication deficient and wouldn't be able to create new viral particles and spread in the unlikely event that you manage to infect your own cells.
Dear Govinda,
Thank you, your answer was enlightening.
One question: when you harvest virus every 12 hours, do you take all of the media from the packaging cells and replace it with fresh media? Or do you take the media in portions? The former would seem logical to me.
Yes, we take it all and then replace it completely with fresh media. You'll want to be very careful at this step so that you don't disturb your cell monolayer.
Hi Davis,
In my lab, we collect the medium on time, about 36h after transduction and we have very good titers (>1E9-1E10 for standard LVs). And we already tranduced hepatocytes without high efficiency, VSV allows the transduction of almost all cells because, as Govinda said, LDL is present in many cells and even its level is low, the high affinity allows the entry of the LVs.
If you need some input of transduction protocoles you can have a look here:
https://www.geg-tech.com/knowledge/lentiviral-vectors-multifaceted-tools/
Hope this will help you
Regards
Nicolas
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