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
- - - - -

Transfection but no expression


  • Please log in to reply
4 replies to this topic

#1 Richard.21

Richard.21

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 38 posts
0
Neutral

Posted 21 October 2013 - 01:38 AM

Hello everybody!

 

I would really appreciate your opinion about this problem I've faced.

I cloned into PcDNA 3.1 Hygromycin and Zeomycin. The insert sequence as well as the promoter regions were sequenced and they're perfect!

I transfect these vector into Mouse embrionic Fibroblast  using Lipofectamine LTX that is quite good for transfect sensitive cells. The transfection rate was stablished at 1:7

Five days after transfection we start the selection.

I ran PCR and the cells were positive as well as they're growing pretty fine under the presence of the highest antibiotic concentration.

On this way that should be expected that these cells would express high levels of my protein but when we screen the cells by FACS they are all negative.

I haven't tested in Western Blotting but anyway they should be positive in FACS.

 

I would really like your comments for this problem

 

Best,


Edited by Cell buster, 21 October 2013 - 04:07 AM.


#2 Tabaluga

Tabaluga

    Making glass out of shards

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 368 posts
48
Excellent

Posted 21 October 2013 - 11:29 AM

Can you still run a WB, just to be on the safe side ? Because i once had the trouble that WB and FACS results were not consistent with each other...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,627 posts
389
Excellent

Posted 22 October 2013 - 12:42 PM

For FACS the antibody needs to be one that recognizes the native epitope - many antibodies will only recognize the denatured epitope - check your antibody datasheet.

 

Do you have a + control for the FACS, - i.e. an antibody that you know works?



#4 Richard.21

Richard.21

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 38 posts
0
Neutral

Posted 22 October 2013 - 11:03 PM

Hi,

 

thanks for your replying.

Yes I have positive controls for FACS that are cells transfected in the past with the same gene. I don´t know what´s going on now that's not working.

I use this antibody: http://www.abdserote...a2461a488t.html

I may have forgotten to mention that these cells are deficient of these genes and we´re trying to rescue them.

 

Kind Regards,



#5 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,627 posts
389
Excellent

Posted 23 October 2013 - 11:59 AM

Two things of note - it is unusual to start selection 5 days after the transfection - by this time many of the cells will have lost the plasmid - growth under selective pressure doesn't mean that the plasmid is inserted!  It could mean that you are using too low a selection level (run a titration before you start transfecting), or it could mean that your antibiotic has gone off, or it could be that the cells are too confluent for the selection to work properly (for most drugs like hygromycin, and zeocin the cells must must be kept below 70% confluent for selection to work). 

 

Checkin for the presence of the insert by PCR may be problematic if the plasmid is not integrated but still present, but Southern blotting  is best - works well for genomic insertion.






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

©1999-2013 Protocol Online, All rights reserved.