Hi,
I want to express a large gene (~10kb) in hard to transfect cells.
I have successfully transduced them with lentivirus, but was wondering what the upper limit was for the length of DNA that can be packaged. Ideally I would want some sort of selection marker as well as my GOI.
If anyone has successfully generated lentivirus that overexpresses a gene of similar size then I would be grateful if you could tell me what vector you cloned your gene into.
Thanks in advance
Nick
Hi Nicholas
I cloned and expressed a fairly large payload in lentivirus, with 16kb between the LTRs.
https://www.ncbi.nlm.nih.gov/m/pubmed/28250438/
Total size of promoter + transgene was about 13kb. I don’t know of any studies involving larger payloads.
In that paper I also investigated rate of titre loss in relation to increasing payload size. Be aware that titre will drop considerably when working with large payloads. This means that the ‘upper limit’ is not necessarily determined by whether it packages or not, but rather whether the size of payload prevents you from achieving a titre necessary for your experiment (obviously very important for in vivo studies).
LV does not seem to have the very strict payload capacity that we see with AAV, where titres fall from a cliff edge when going above 4.8kb.
When making LV with large payloads, you should consider modifying your lentivirus production methodology by transfecting an equal molarity of plasmid, rather than basing transfection amounts on mass in micrograms (as per conventional protocols). This should help maintain a decent level of vector RNA.
Best
John
Thanks for your answer John,
It's good to know that it is possible. By my calculations the payload for my virus should be ~15kb.
That's a useful tip about adjusting amounts for the increased size of the vector.
Cheers
Nick
- Badges
- Science method