I need to clone kanamycin resistance gene into a eukaryotic vector. Can I amplify the kanamycin resistant gene in pET28a(+) vector and clone it into the eukaryotic vector? Will it confer neomycin resistance when I transfer it into cells?
It's not as simple as just cloning the gene, you also need to engineer it for appropriate expression in eukaryotic cells, such attaching a promoter and translational signals. The bacterial promoter will not drive expression. Also you need to use G418 and not neomycin for selection in eukaryotes. It might be easier to begin with a construct already designed for eukaryotic selection, there are many out there.
@Michael J. Benedik . Thank you for your suggestions prof. I am planning to put in after a CMV promoter. Will that be enough?
The kanamycin resistance gene from pET-28a can be digested with specific restriction enzymes and cloned in a eukaryotic expression vector if it contains same restriction sites. Or it can be amplified with high fidelity polymerases (eg. pfu polymerase) and cloned in to your vector of interest. For the proper expression kanamycin resistance gene it should not contain any mutation (which may occur during PCR amplification) and should have a promoter (eg. CaMV 35S) and a terminator sequence. If the gene expresses properly, the transferred cells will confer kanamycin/neomycin resistance. Best wishes.
Santanu Sasidharan ,
The aminoglycoside phosphotransferase (KanR) gene of pET28a+ comes from Tn903, and is different from the aminoglycoside phosphotransferase, of, say, the pcDNA3 family of vectors, which comes from Tn5 and does confer resistance to kanamycin, neomycin and G418. In other words, there is a possibility it might not work for your intended purpose (substrate specificity might differ, for instance).
If I were you I'd follow Michael J. Benedik 's advice and simply borrow the NeoR cassette from an existing eukaryotic expression vector.
Hope it helps!
Santanu Sasidharan ,
A colleague mentioned over lunch that she was pretty sure the KmR gene of Tn903 does confer resistance to neomycin and G418, at least in plant cells, so I searched a bit as soon as I had a chance. It seems she is right:
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Geneticin_UG.pdf
- Badges
- Science topic
- Similar topics
- Biological Science
- Biota
- Eukaryota