To get around 4 kb, would any non-coding sequence be ok? Some use parts of lacz, and some from the pBR322 plasmid etc. Maybe someone has some sequence from the lacz gene (we have this in our lab) that they know would work as a stuffer sequence?
Hi Josephine,
It is not necessary to include a stuffer sequence, more cloning, but the titers will not be much higher. Our vectors range from 2,000 to a bit less than 5,000 between ITRs and there is no large differences in the titers. Check these two papers about capacity. The older one shows that between 3,000 and 4,700 the difference is max 1,5x
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19904234
http://www.ncbi.nlm.nih.gov/pubmed/8934224
Hi Josephine,
It is not necessary to include a stuffer sequence, more cloning, but the titers will not be much higher. Our vectors range from 2,000 to a bit less than 5,000 between ITRs and there is no large differences in the titers. Check these two papers about capacity. The older one shows that between 3,000 and 4,700 the difference is max 1,5x
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19904234
http://www.ncbi.nlm.nih.gov/pubmed/8934224
OK, thanks for your response. :)
We will still do this, because we have one vector contains GFP and one that expresses our gene of interest, and we want everything to be as equal as possible (although that is hardly equal, while expressing a foreign gene like GFP..), as the plan is to take this to clinical trials later. :)
All that said, you never design stuffers then?
Thank you,
Josephine
No, we do not include stuffers. I think that any bacterial plasmid backbone (like pBR or pBS) will work well if you put it between polyA of your GOI and 3' ITR.
Thanks for your answer, we were just a bit confused as there is not much in the literature discussing these things. :)
Thanks, Konstantin!
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