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Gateway cassette and IRES site

Gateway IRES attR GFP

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#1 Glis

Glis

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Posted 09 October 2014 - 05:07 AM

Dear all,

 

our lab bought the plasmid pLVX-IRES-Neo and I should make it Gateway-compatible, as well as replace the Neomycin gene with the GFP gene. The replacement of the genes was no problem and after some hard weeks, I finally ligated the attR-ccdB-CmR-sequence into the MCS of the pLVX-IRES-GFP plasmid (CMV promotor - attR1 - ccdB - CmR - attR2 - IRES site - GFP). I did some LR reactions and made test transfections with the received expression plasmids to check if the GFP is working.

 

Unfortunately, I can't see (via fluorescent microscopy) or detect (via FACS analysis) any GFP signal. Neither with the empty plasmid pLVX-attR1-ccdB-CmR-attR2-IRES-GFP, nor with my expression plasmids pLVX-attR1-cDNA-attR2-IRES-GFP (my positive control - a GFP expression plasmid - however is fine).

 

What could be the reason why I can't see any GFP signal in my transfected cells? 1) Is the CMV promotor too weak? 2) Do the Gateway cassette and the IRES site probably "not work together" (I searched the web, but I didn't find expression plasmids with the Gateway cassette AND the IRES site)? 3) Should the (start codon of the) GFP gene be in frame with the TAC AAA sequence of the attR2 site (like mentioned in the Gateway manual)? 4) Or is anything else the problem?

 

Thanks for your help,

Glis







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