If you insert, after the promoter at the site of the Ampicilline-ORF, an EGFP-ORF and you transfects this plasmid into mammalian cell line, do you see green fluorescence after 48 hours?
Hi,
In most plasmid vectors Ampicillin resistance gene (beta lactamase) is expressed under the control of P3 promoter, which to the best of my knowledge is not active in mammalian cells. Therefore, EGFP gene cloned downstream P3 promoter should not get expressed in mammalian cells. Unless you are using a vector in which beta lactamase is expressed under the control of a promoter (such as SV40 or CMV etc.) active in mammalian cells. Which plasmid vector are you using?
Thank you for your answer! It's a question from my school, so I actually don't know which plasmid vector it is. But in my practicum I make use of the plasmid pcDNA3.1, and this one consist of a CMV promoter and SV40. But this plasmid also contains the neomycine gene, which is more for mammalian cells. So if the Ampicilline-ORF can be expressed in mammalian cells, then you would see after 48 hours fluorescence, but if not then you wouldn't see something?
There are two selection markers on pCDNA3.1 vector. One is Ampicillin resistance gene and other is Neomycin resistance gene.
Ampicillin resistance gene is expressed under its native P3 promoter and in bacteria. The purpose of this selection marker is to propagate the pCDNA3.1 in bacterial hosts that are used for cloning and propagation purposes.
The Neomycin resistance gene is expressed under SV40 promoter and in mammalian cells. Neomycin resistance gene is used for stable transfection where mammalian cells are cultured in presence of antibiotic G418. All untransfected cells get killed whereas those containing pCDNA3.1 (and constitutively expressing Neomycin resistance gene) survive.
So back to your original question; if you replace Ampicillin resistance gene ORF with EGFP, it wont express in mammalian cells but in bacteria. If you replace Neomycin resistance gene ORF with EGFP, it will express in mammalian cells but not in bacteria.
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