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No expression in stable transfected cell line


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#1 Morrigan

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Posted 26 March 2014 - 06:13 AM

Hello everyone,

 

I have a problem with my stable transfected myoblast cell line. I used a lentiviral vector for transduction and selected the cells with puromycin for about a week until all untransduced cells in the control plate had died. Prior to transduction a kill curve was performed with this cell line to determine the appropriate amount of antibiotic. In the western blot analysis of the cells, after selection, I couldn't detect my protein of interest. I also performed a reverse transcriptase PCR and the construct seems to be expressed on mRNA level. Now I'm wondering why there is no expression of the protein.

 

I hope you can help me out with some suggestions or advice on this problem. Also, if you know any useful literature concerning this topic, I'd really appreciate if you'd share it here.

 

Thanks a lot!



#2 Rajpoot

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Posted 26 March 2014 - 09:19 PM

Did you put kozak sequence before start codon of your ORF?



#3 Morrigan

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Posted 27 March 2014 - 12:49 AM

It doesn't seem to be a problem of the plasmid. I transiently transfected the construct in HEK293 and could detect the protein afterwards. It's only missing in the stable transfected cell line.






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