pcDNA 3.1/pTarget and stable cell line - (Nov/24/2005 )
I have constructs in pcDNA 3.1 (Invitrogen) and in pTarget (Promega). They are proteins that assemble together in the cell membrane. I want to do transfection with both of them using Lipofectamine 2000 (invitrogen), do you think I will have any problem?
More important, could I get a stable cell line with both constructs? Or on the contrary, this cell line may not last more than 6 or 8 passages and then I will get deterioration, what do you think?
I would appreciate it if anyone could give me some advice, potential problems, etc.
hi
double transfection is not a problem as you would use equal mass of each plasmid and total weight do not exceed the appropriate weight you use for single plasmid transfection
but in your case... well if i'm not wrong both of these vectors are carrying the NeoR gene?
so you will select both double transfected and single transfected cells... so you need to have 2different selection markers to generate double transfected cells...
an other possibility : to do clones after the transfection and test each one for presence of 1 or 2 plasmids. But you may have to screen many clones to get your one
You are right (as usual).
G418 can be used for pc3.1 and probably will make the same effect in pTarget, am I right?
Do you now what different selection markers I could use?
Resistance of the cells to G418 will come from the expression in the cell of the Neo from pCDNA or pTarget. So you won't be able to say between 1 or 2 plasmids in resistance cell.
Selectable markers :
puromycine, blasticidin, hygromycin, zeocin, phleocin, or plasmocin.
honnestly the more comfortable is puromycin. Just add puromycin and positive cells only in 1 week
you cannot go wrong...i would keep pTarget which seems more user friendly as pCDNA.
puromycine, blasticidin, hygromycin, zeocin, phleocin, or plasmocin.
honnestly the more comfortable is puromycin. Just add puromycin and positive cells only in 1 week
you cannot go wrong...i would keep pTarget which seems more user friendly as pCDNA.
Well, when I said the same effect I meant that I would be unable to separate two plasmids colonies from one plasmids colonies.
Standard pTarget and pcDNA 3.1 are only resistant to G418, isn't it?
So to make my plasmids resistant to different selective markers then I also need to insert one resistant sequences into each plasmid for make one resistant to, for example, puromycin, and the other to blasticidin. Is this the only solution?
The pTarget and pcDNA 3.1 that I got have inserted different sequecences and I need express both at the same time. What did you mean with keep pTarget?
Sorry if I look a little lost (in fact, I'm quite)
Thanks
well, yes. as both plasmids contains Neo R gene, you need to use for one plasmid Neo and for the other an other selective marker. That's why i sais one plasmid can be NeoR and the other puro R for ex. I recommended puro as it's the easiest selection i know.
i've understood only now that you've already inserted the two cDNA you want to express.
If you didn't have done it i said keep pTARGET as he presence of lacZ sequence allows easy recognition of recombinant plasmids.
As both vectors contains insert, you may keep the more useful one for you.
I worked few month ago on a plasmid and i've changed neo R gene for puro R gene by simple digestion ligation between two plasmids,, first with neoR gene, and second with puro gene.
So i would recommend you to do the same.
Great, thanks a lot for sorting out my problem.
I will do the same you did.
May I ask you which was the plasmid with puro R that you used? Is it suitable for Mammalian expression?
"i've changed neo R gene for puro R gene by simple digestion ligation between two plasmids,, first with neoR gene, and second with puro gene"
Doing this you take the insert from one and you inset into the second plasmid, isn't it? You change the whole plasmid, not only the Neo R gene, am I right? Because I was looking for restriction sites within pTarget to cut only the puro R gene but you need to take everything except from the inserted sequence.
Thank you one more time.
hi
i took the puro gene from pSUPER between BamHI and BstBI taking both puro orf and PGKpromoter. I cut the target plasmid xith BstBI and BclI (BclI dam sensitive and BamHI compatible) making target plasmid puro resistant (i just cut the Neo gene of it and replace by PGKpuro).
pSUPER puro is not actually a mammalian expression plasmid. But i'm gonna use in a next experiment the pBABEpuro vector which is actually suitable for mammalian expression. You can find map of it by googling or here at stewart lab webpage.
Hi
Thank you one more time and sorry to bother you again.
I’ve been checking your strategy and yes, it’s fine if they are no mammalian vectors.
My problem is that I’ve done a restriction sites map of pTarget and pBABE puro and yes, BamHI is the best one for upstream in pTarget and pBABE, but I can’t find any suitable for downstream (3581 in advance). It doesn’t seem that pTarget has got many restriction sites to cut the Neomycin marker.
Neomycin selectable marker (including SV40) is from 2260 to 3581. The only restriction sites which cut above 3581 are EcoO109I(3786) and PfoI(3727) because others also cut into B-lactamase coding region. As you can imagine, these two don’t cut only once the pBABE puro vector.
May I be mistaken? Should I look for another puro vector or insert restriction sites using primers?
If you are gonna use pBABE, can you tell me the strategy to follow? (I hope it will not be confidential because I’m a little bit lose again).
I would be really grateful if you help me with this last step (I hope it will be the last one). I
Thank you very much.
hi
in your case, easiest and i think quickest method too is to PCR the puro gene and ading Two restrictions sites more "user friendly" for your plasmid.
what do you want to know about pBABE? i didn't get pgk puro from it...
you mention that you want to do a pTARGET puro but isn't it easier to do that in pCDNA?...