5 replies to this topic

[Discuss]

Is it possible to use a lentiviral based vector/plasmid for other transfection methods eg. CaCl2 method?

[PostDocTrauma]

you don't need to transfect a lentivirus. Just put an aliquot on your cells and they will be infected.

[Curtis]

I think this vector was made when the CaCl2 method was already old fashioned. I haven't seen any protocol for lenti vectors and CaCl2. But you can try. I never had problem with CaCl2 transfection when I did it during my MSc, however most people find it inefficient.

[Discuss]

Thank you so much for the replies.  I asked the doubt because I have a vector which contains a HIV LTR (for lentiviral transduction) as well as a CMV promoter. I know that it has been previously used for lentiviral transduction of mammalian cells but since I do not want to use that method and since the vector is already available for me,  I was wondering if it is possible to use it for a ‘non-lentiviral’ method.  Also in general, if a vector has two promoters, can it be used for expression of a single gene? I have used vectors with single promoters before to express genes, but this vector has more than one promoter. Anyone has any comments?

[dpo]

Yes, you can simply transfect the plasmid that encodes the viral genome in any cell. Most of the time (depending on the specifics of the lentiviral vector you are using), this will result in the expression of your gene of interest. I do this all the time to try out my constructs before making virus of them.

I'm not really sure what to answer on your question on the two promoters. Where are these located? For instance, if one promoter is located upstream your gene of interest and the second one in front of the antibiotic resistance gene, then it's no problem to use this vector for the expression of your gene of interest.

[Discuss]

That was so helpful!!! I got the answer.

dpo, on 10 August 2011 - 05:01 AM, said:

Yes, you can simply transfect the plasmid that encodes the viral genome in any cell. Most of the time (depending on the specifics of the lentiviral vector you are using), this will result in the expression of your gene of interest. I do this all the time to try out my constructs before making virus of them.

I'm not really sure what to answer on your question on the two promoters. Where are these located? For instance, if one promoter is located upstream your gene of interest and the second one in front of the antibiotic resistance gene, then it's no problem to use this vector for the expression of your gene of interest.