why a low copy number? - shRNA vector (Feb/06/2008 )
So the commercially available vector that I have subcloned my shRNA into is a very low copy number vector. Why, in this age where we can pick which ever ori we wish, is it important that this vector be low copy number? I know that it is possible to increase the copy number by spiking my broth with chloramphenicol. Is that an advisable thing to do? I ask because on the one hand I need sufficient plasmid to make virus, but on the other hand if there is a reason that the vector is a low copy number vector then maybe adding the chloramphicol will all the R&D that went into making the vector.
Also, I'm new to shRNA, could someone explain to me what all the buzz is about with chosing a rec- bacteria to grow up the plasmid in? Why is recombination such an issue to shRNA plasmids? And if recombination can occur should I get my plasmid sequence after doing a maxiprepp (or from my initial miniprep )?? If this plasmid says on its spec sheet that I can grow it up in regular DH5alpha, then do I still need to be worried about recombination??
Thank you in advance!!
for just cloning shrna into a plasmid the rec- is not so important, same with low copy number ori. The suggestions are, correct me if i'm wrong, mainly for cloning shrna into retro- or lentiviral vectors because the recombination frequency between the long terminal repeat (LTRs) is quite high. Low copy number also seems to help against recombination events. Introducting a complementary sequence like shrna between the virus LTRs pushes up the recombination frequency even more it seems. So for cloning shrna into a non viral plasmid is a lot easier than cloning into a viral one. I never did chloramphenicol amplification myself, i just increase the culture size (500 ml instead of 300 ml for maxi prep). I always do a control digest of new maxi preps to see if the plasmid is still ok. The first maxiprep i always sequence it. This is very important for shrna sequences especially if you get the sequence synthesiszed as ss DNA oligos or generate them by PCR.
Many viral vectors have terminal repeats which can undergo recombination and actually modify the vector. i have seen cases where the TR's are missing on one side and you donot get virus production. The same for shRNA. Its better to have them in rec- cells. We verify plasmids after maxipreps.
I would be worried if this vector has terminal repeats even if the spec sheet says it can grow in DH-5alpha.
To overcome the problem of low copy, we usually do a 10ml day culture and use it to inoculate a 250 ml or 500ml culture depending on how much you need. We get about 1-1.5 mgs of DNA.
Thank you ! I appreciate your comments.