Hi, I am quite new to the plasmid selection guide. I wish to select a GFP plasmid for lentiviral assembly to finally transfect mouse cell, and also for an another study: shSOD1 expression plasmid having any selectable marker (eg. GFP/mCherry/Puro etc) for mouse cell. When i tried to look for a mGFP expression vector, it gave so many choices that I was unable to find the suitable one for me. Please guide regarding the same.
Thanks in Advance.
syed
Your target cell is important for promoter choice. Then the question if you want it stable or transient. So more information will give more precise answers.
Dear Syed,
the best vector for your needs probably must be bicistronic, better with two different promoter avoiding IRES. In my hand one of best letnvirial vector I ever used is the TWEEN with CMV ....PGK-GFP, thus you can use it for cell labeling and to subclone shSOD1. Anyway you cna check into clontech site for other GFP vector or as suggeste by Li into Addgene web site.
I have used bicistronic and monocistronic systems and I would always go for monociistronic but with a T2A side instead of Ires. This gives you security that every cell expressing your marker will express your transgen
Regars Svven
No Stop codon ths is right. I am using the system bioscience vecttor set but also modified them with other selection markers. In case you want to do it by PCR you can design it in a kind of overlap extension PCR but 100bp Primer works also good
Regards Sven
Just to complete the answers about 2A sequence.
There is a paper which compare the different sequences 2A and the authors conclude that P2A is the best:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018556
But I presume that it is depending of the experimental context
Nicolas
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