Is important the direction of IRES-GFP in cloning? - (Sep/21/2014 )

Dear respected forums
Hi ,

 I am trying to insert IRES-GFP into pCMV-SB100 vector and for it , I use of one ApaI site on this vector (one enzyme cloning), right after SB100 ORF . I did PCR IRES-GFP region from plEGO-IG vector and cloned that PCR product into SB100 vector to drive GFP expression by the CMV promoter, which already drives SB100 expression. I got a few clones which have the IRES-GFP insert (non-directed) and in this week I'm going to directly transfect it into 293T cells .
I'm writing to ask you , is important the direction of  IRES-GFP for my project?  Because final construct involved Promoter(CMV)+ SB100+GFP+IRES+PA terminator , I think that clones will show GFP expression however maybe GFP  not strong compared to GFP in pLEGO-IG vector.



 
Thanks a lot in advance.

- Mahdi-

esmaeilzadeh on Sun Sep 21 07:52:26 2014 said:

Dear respected forums
Hi ,

 I am trying to insert IRES-GFP into pCMV-SB100 vector and for it , I use of one ApaI site on this vector (one enzyme cloning), right after SB100 ORF . I did PCR IRES-GFP region from plEGO-IG vector and cloned that PCR product into SB100 vector to drive GFP expression by the CMV promoter, which already drives SB100 expression. I got a few clones which have the IRES-GFP insert (non-directed) and in this week I'm going to directly transfect it into 293T cells .
I'm writing to ask you , is important the direction of  IRES-GFP for my project?  Because final construct involved Promoter(CMV)+ SB100+GFP+IRES+PA terminator , I think that clones will show GFP expression however maybe GFP  not strong compared to GFP in pLEGO-IG vector.



 
Thanks a lot in advance.

- Mahdi-

If you mean to say that the IRES-GFP went into the vector in the opposite orientation, I would think it would make a pretty big difference.  Your RNA sequence will be transcribed under the CMV promoter but the sequence as read by the ribosome (if indeed it recognized the antisense IRES) would be the antisense strand.  You'd need the IRES and GFP to be in the same orientation as the rest of the RNA being transcribed.  (I hope I understood your question correctly).