(no subject)/macs and CATs

brett brett at BORCIM.WUSTL.EDU
Thu Dec 14 14:31:22 EST 1995


>Hello together,
>does anybody have experience with the CAT reporter system provided by 
>PROMEGA (pCAT-Basic, -Control, -Promoter, -Enhancer)? We made a lot of 
>experiments trying to introduce pCAT-Control into murine or human 
>macrophage cellines (RAW246 and THP-1), only to determine the best 
>transfection conditions. But up to now we weren't able to introduce 
>anything. We tried DEAE-Dextrane and electroporation in many different 
>variations - ever without any success!!!
>I'm beginning to doubt about me. It must be possible, to get this vector 
>in the cells. Or is anybody wrong with the control plasmid?
>The CAT assay itself is ok because we are making control experiments with 
>pure CAT enzyme.
>
>Has anybody made similar experiences?
>In advance to your answer, best regards
>
>Dirk Seegert, Ph.D.
>Fraunhofer Institute Hannover, Germany

I think you should check your plasmid in an easily tranfected cell, such as
NIH-3T3, L929, BHK-21, or HeLa. Then work out conditions for macrophages.
Dig up the ongoing thread about transfecting macrophages. I have used
a CaPO4 technique for stably transfecting RAWs, but have not tried transient
expression. Also, there is someone in my lab, who I can put you in touch with,
that routinely transfects THP-1 cells transiently with DEAE-dextran. In either
case, expect low efficiency. That means, scrap the CAT assay and switch to
luciferase. It is much more sensitive. 


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




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