I am wondering whether the length of the insert cloned in a lentivector would effect the packaging efficiency or the viral titer? Did any one notice any such changes?
Absolutely. There is a huge effect of the size of the insert vs titers.The longer the insert, the less virus you'll get. I have gotten virus to be produced from lentiviral vectors that are 12.5-13 kb in size (the size of the actual lenti, from LTR to LTR is then little over 10k). But titers are really low as compared to lentis with a vector size of ±9kb (lenti genome size of 6-7kb), several log that is. It does make sense, there is a limit of what size RNA you can fit into the virion...
To have efficient packaging, the region between the 5’and 3’LTR must not exceed 9kb, which is the size of the native HIV genome. To calculate the size of the insert that the lentivector can accommodate, download the vector sequence and determine the size of this region. The difference between 9kb and the size of the 5’- to 3’-LTR is the limit of the size of the insert for optimum packaging.
Hi Afzal,
The link between size of genome and titer is very well describe in this article:
http://online.liebertpub.com/doi/abs/10.1089/104303401753153947
If you don't have the access to this paper i can send you it by email if you want.
Nicolas
Hi Nicolas,
Could you please send me the article? My email address is [email protected]
Hey guys,
It seems to be of common sense that the viral titers drop incredibly with larger vectors.. but has anyone actually tried with inserts > 13kb ( from 5' to 3' LTR + cloned insert between the LRT regions) ? Could I have a minimum workable virus tritation?
Robert Jan Lebbink you said you had 12.5-13 kb inserts, but beyond 5'-3' LTRs, right?
Thanks a lot
I have tried 6.3 kb as an insert and the total vector size became 16kb. the titer dramatically decreased, so I made a few optimizations to enhance the titer. It worked but still not reach about >10^7 virus
It does affect, but I got good results packing a 13kb 5-3'UTR lentiviral plasmid.
Hi Rajiv, I use the viral supernatant to transfect colon cells and then I make puromycin selection ( the viral plasmid contain puromycin resistance).
Ahha. Thank you for your reaction.
I thought you might have determined
the number of infectious units or virus particles/plasmid copy number
Because obtaining a lower titer (infectious units) using a bigger plasmid could also be the consequence of a lower transfection efficiency of a bigger plasmid into the virus production cell line.
Hi Yasmin, what is the transduction efficiency(with a GFP or other fluorescent protein)? I am trying to transduce a total 13kb (insert+backbone) and efficiency is low. How did you improve the titre? Thank you. @Yasmin Lima
Hi Roberto Avellino I didn't use GFP, but puromycin selections instead, sometimes my efficiency was not that high, but the selected cells were enough to then propagate.
Roberto Avellino I couldnt obtained infected cells with MOI greater than 1 though
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