Hello:
We are using a lentiviral plasmid - pLVx-EFalpha1-AcGFP-N1 and we cloned a very large protein (about 9000bp) and a smaller protein (about 500bp) into the plasmid. We used EcoR1 for the cloning, this restriction enzyme site is not identified on the MCS by Clontech but someone in my lab found it and it seemed to work. We had no problems with the sub cloning and sequences both and they look fine. However, when we try to make virus, we do not get any GFP expression. We checked for virus using a GoStix and there was virus but unclear if any of our plasmid was in there. We then transfected HEKs with the plasmids, the empty vector did have GFP expression and our protein in a different vector had GFP expression but any protein in the PLVx vector did not have GFP expression. We called the company and they said they had never heard of EcoR1 being a problem before. We are wondering if anyone else has had a similar experience?
This vector does, indeed, have a unique EcoR1 site, so I doubt that EcoR1 cloning is the problem. If you cloned EcoR1-EcoR1 you may have contamination with recirculaised empty vector after ligation (did you dephosphorylate before ligation ?). Even if the concentration of empty vector is low, the empty vector is small and will be positively selected during viral production.
Packaging might be inefficient for the larger of your two constructions, but this should not be the case for the smaller construction.
Have you selected your transformed cells with puromycin to ensure that you have had infection/integration ?
This vector does not have an IRES so you will have to make sure that the GFP sequence is in-frame with the sequence coding your protein of interest.
good luck with the expts
Hi Jessica Tsui, I agree with Ian that EcoR1 is not the problem as our Lenti vector got 4 EcoR1 sites and we got great GFP expression in both tranfected and transduced lenti.
Do you have an postive control lenti vector that you could test (the lenti vector with only GFP and compare to your Lenti vect with GFP and your insert)?
All the best
Hello:
Thanks all for your help. We do have a positive control vector which also did not work when making virus but did work when we just transfected it using TransIT-293 into HEK cells. We did check the sequences of our vectors + insert and it looks like the GFP sequence is in-frame. Now we are thinking that it may be a problem with our Maxi-Prep - we do not use a column and instead use another protocol with chloroform and phenol/chloroform which increases our yield but we are thinking that there may be something that is binding to the DNA that is preventing the DNA from complexing with the calcium phosphate when we try make virus. We are trying a new Maxi-Prep with a Qiagen column to see if that takes care of it. Thanks again for all of the input!
Ian Robbins has said it all.
Dear Jessica First, if none of the things mentioned above are the case, are you sure the vector has not been subjected to mislabeling so you would use the wrong plasmid?, Things happen
Second, pure DNA is a must in virus production and transfection.I am not saying it is not possible instead I say it is possible to produce virus even with impure DNA, but the point is highly expensive experiments do not deserve to fail because of the impure plasmids.
Third, any contamination of chloroform/phenol may inhibit restriction enzymes and transfection.
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