I want to construct plasmid for mammalian cell transfection with cDNA from adenoviral vector(pAdRFP).
But my instructor said, if I use cDNA from adenoviral vector, there is risk for contamination of viral DNA component. but the restriction sites are fit well with cDNA sequence. There are no UTRs at 5' and 3' of the ORF.
Is it really possible? I think it doesn't matter, but I need more advices from others.
Thank you.
I'm slightly confused about your question - pAdRFP does NOT contain a full adenoviral genome. It is simply a plasmid that contains your gene of interest plus the necessary elements so that when you co-transform this (shuttle) vector and an adenoviral backbone plasmid vector, pAdEasy- into E.coli, then you get adenovirus genome containing your gene of interest. You would then need to transfect this into HEK293 cells to get adenovirus particles
https://www.addgene.org/12520/
https://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0ahUKEwiy4-j19YHYAhWiA8AKHZJ2CisQFggpMAA&url=https%3A%2F%2Fwww.agilent.com%2Fcs%2Flibrary%2Fusermanuals%2Fpublic%2F240009.pdf&usg=AOvVaw0FKpPk6M0p56sNdkgZ3ZXS
The use of the word cDNA suggests that you intend to clone DNA you made from RNA, when the vector=plasmid is already DNA... if you are suggesting to make cDNA from adenovirus then again I'm confused as adenoviruses have a double-stranded DNA genome - no RNA involved, unless you are attempting to measure gene expression, but then I'm not sure why you want to clone into pAdRFP.
It seems to me, that the language barrier is preventing you from describing your problem in a way we can understand...
I think your question is - Can I use pAdRFP as an expression vector to express my gene of interest in mammalian cells?
I would recommend reading this http://info.addgene.org/plasmids-101-topic-page
and that should help you understand what you are doing and if pAdRFP has the elements it needs to get expression of your gene of interest in mammalian cells.
If you are still stuck in a few days (after reading the booklet!) then message me and I'll give you the answer. But I'd really like you to try understanding first.
Thank you for your answer. Please understand that English is not my mother tongue, so some expressions may be confused.
My question is, 'Is it okay to use gene of interest from pAdRFP vector without problem?'
My instructor worries about the problem, if the constructed pcDNA3.1 plasmid is contaminated with very small particle of viral genome. (Actually he is sensitive to this kind of problem. Quite! )
I just cut the gene of interest from the pAdRFP vector, and ligate it into the pcDNA3.1.
But according to your answer, pAdRFP is just a plasmid vector, not containing full sequence of adenovirus, right? So is it okay to use the constructed pcDNA3.1-My gene of interest plasmid?
The pAdRFP described by Addgene is just a normal plasmid - but make sure that it is the same plasmid as what you have in your lab by comparing the maps/sequences.
If you prepped pAdRFP in E.coli without the second plasmid containing the adenoviral backbone (pAdEasy) then no contamination possible.
pAdRFP on it's own does NOT contain the adenoviral genome, therefore no contamination.
If you mean that you want to use E.coli plasmid prep as a template where you had co-transfected pAdRFP and the adenovirus plasmid (pAdEasy) then there still wouldn't be contamination - the two plasmids are meant to recombine into one big plasmid which contains the adenoviral genome (but NOT viral particles until you transfect into mammalian cells). If you cut this DNA with a restriction enzyme and then gel-purify your fragment then you will only ligate your fragment into the pcDNA vector.
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