I want to express a human protein in mammalian cells. Why it is always advised to first clone (amplify) it in bacteria and then use those cloned plasmids for expression in mammalian cells ? Can't we directly transform the mammalian cells inspite of first cloning it in bacterial cells and then using those plasmids to transform mammalian cells in order to express the gene of interest as mammalian cells will also clone (amplify) it as they replicate and then express it ?
Dear Shubham,
This will allow you to amplify/synthesize more plasmid if you use bacteria
Yes, you will get more plasmid and it will have less pitfalls sub cloning in prokaryotic cells, vs more complex eukaryotic cell. If your final protein is eukaryotic it may not fold correct in bacterium and need euky translation for activity
Mammalian cells will not replicate your plasmid. The cells will slowly eliminate the plasmid unless it is integrated into the genome (stable transfection).
Hi,
It is easy to handle bacteria and it divides every 20 minutes which means you can get a lot of your required DNA just by growing them overnight!!!
I agree with all the above comments. I only add that it is easy and cheap to do the cloning part in E. coli. Once you have the correct clone, you express it in mammalian or other systems.
The plasmid can copy indeplimently in bacteria，but can't in mammalian cell，you must get enough plasmid in bacteria first and then transfer it to mammalian cells for protain。
I totally agree with Elizabeth. Plamid is a natural component in most bacteria, but not in mammalian cells. You have to amplify it in bacteria instead of mammalian cells.
I am using shuttle vector having ori for bacteria as well as SV40 ori for mammalian cells. Howcome, it will not replicate in mammalian cells then ?
Elizabeth, it would be very helpful you could explain your statement because according to your answer, even if we transfect mammalian cells after amplifying the plasmid in bacteria, it will eventually eliminate all the plasmids.
The SV40 origin will only be active in cells that express the SV40 large T antigen which recruit the cellular replication machinery to the viral origin.
As Elizabeth pointed transiently transfected plasmids will get diluted as the cells divide until they are gone, hence transient.
On a technical note, doing all the steps you need to do when cloning such as screen for positive clones, extract the plasmid in order to get it sequenced, and amplifying the verified clones will be extremely laborious and time consuming turning a process that, using bacteria, will be easily done in under a week into a mounts long ordeal with an infinitesimally small chance of success.
Yoav gave you the answer to the question you addressed to me. I have been working in academic research labs since 1979 and I have learned that it is most important (especially with kits) to know why you are doing what you are doing. Read, read, read.
As an example, how many people know how an RNA extraction kit works?
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