Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

High copy vs low copy plasmids: which one causes less toxicity?


  • Please log in to reply
2 replies to this topic

#1 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,198 posts
63
Excellent

Posted 12 January 2019 - 11:16 AM

I recently got one of my review articles rejected. One of the reviewers made some comments about a paragraph that I had. Do you agree with him? By practice we have found that if we use hich copy plasmids for recovery of virus then more cells die. The reviewer says that this does not depend on the copy number, but the promoter. We used the same T7 promoter for both plasmids. The low copy ones were less toxic.

 

Paragraph 2.1 contains a number of statements that are not correct. The authors argue that the use of high-copy number vectors has "... the advantage that they result in high transcription rate". This is not true; the transcription rate in eukaryotic cells is primarily determined by the promoter, not by the plasmid copy number in E.coli. Also the statement that "... low-copy number transcription vectors usually produce less toxicity in cell culture compared to high-copy number vectors ..." is not true. The choice between a low- or high-copy number plasmid is primarily one of convenience (higher yield) or plasmid stability in E.coli.



#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,592 posts
559
Excellent

Posted 14 January 2019 - 08:57 AM

The reviewer is quite correct - the T7 promoter is a prokaryotic promoter that doesn't result in expression in eukaryotes, so high copy-number vs low copy in eukaryotes shouldn't be dependent on this promoter. In addition, the copy number is prokaryotes is controlled by the ori, and is not related to the level of RNA expression. I think you will find that your plasmids contain another promoter system as well: probably SV-40 or CMV, which are used to induce high levels of RNA in your cells and result in different levels of RNA being expressed. The eukaryotic viral promoters are very high expression, but you can get low expression plasmids, which usually have mammalian promoters such as K14. Often these promoters are used to target expression to particular cell types - K14 is a keratinocyte specific promoter.

 

Most plasmids do not replicate in cells either - there are a few exceptions such as ones that contain EBNA, which can be used to replicate the plasmid in the cell (outside the nucleus I think).  

 

 

I have found that high copy plasmids tend to carry more bacterial proteins etc over with them when doing purification, these can be cytotoxic to eukaryotic cells.



#3 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,378 posts
136
Excellent

Posted 20 January 2019 - 01:22 AM

Bob1: What about cells with SV40 large T-antigen, like HEK293T? Those are supposed to replicate plasmids with SV40 ori, due to viral-based mechanism and thus increase transcription as well. 
AFAIK immortalization with SV40 large T-ag is quite common now.

 

Curtis: What cell line you have and what plasmid backbone? Many plasmids have SV40 ori.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.