Using IRES in microRNA expression vector - (Jul/01/2008 )
Hi,
I am planning to make of series of lentivirus constructs to stably express microRNAs in cell lines. My expression cassette is in this order:
CMVp-premiR-IRES-mCherry
My question is would processing of the premiR interfere with downstream translation initiation from the IRES?
I have looked at the maps of commercial vectors or those in publications and usually, the order is:
CMVp-GFP-premiR without any IRES between GFP and premiR.
Any input would be appreciated. Thanks 
Hi,
I have done a construct CMV-premiR(~400bp)-DsRed and did the transfection once. I didn't see any fluorescence. Because it was preliminary I didn't check for the expression of miRNA. The company SBI has actually two promoter for the premiR and reporter. (http://www.systembio.com/miRNA_Precursor_Collection.htm)
There could be some issue with the IRES unless the resulting mRNA is still unprocessed.
Gunter
I have done a construct CMV-premiR(~400bp)-DsRed and did the transfection once. I didn't see any fluorescence. Because it was preliminary I didn't check for the expression of miRNA. The company SBI has actually two promoter for the premiR and reporter. (http://www.systembio.com/miRNA_Precursor_Collection.htm)
There could be some issue with the IRES unless the resulting mRNA is still unprocessed.
Gunter
Thanks Gunter. I have looked at the SBI vector. The copGFP and premirR will theoretically be on different transcripts so there would be no interference.
I will give my constructs a quick try by just transfecting them into cells to look for fluorescence. I have already started to make the constructs when it just occurred to me to check. Anyway, FYI, there is one paper that describes a construct as follows:
MSCV-miR-203-IRES–GFP
Hope they are right!
Cheers.
MAC
I have done a construct CMV-premiR(~400bp)-DsRed and did the transfection once. I didn't see any fluorescence. Because it was preliminary I didn't check for the expression of miRNA. The company SBI has actually two promoter for the premiR and reporter. (http://www.systembio.com/miRNA_Precursor_Collection.htm)
There could be some issue with the IRES unless the resulting mRNA is still unprocessed.
Gunter
Thanks Gunter. I have looked at the SBI vector. The copGFP and premirR will theoretically be on different transcripts so there would be no interference.
I will give my constructs a quick try by just transfecting them into cells to look for fluorescence. I have already started to make the constructs when it just occurred to me to check. Anyway, FYI, there is one paper that describes a construct as follows:
MSCV-miR-203-IRES–GFP
Hope they are right!
Cheers.
MAC
I have tested my constructs and the mCherry was not expressed! It seems like pre-miR has interfered with the processing of the transcript. Now, I'll have to cut out the IRES and substitute it with a promoter, making something like the SBI vector. Has anyone else out there faced the same problem?
Thanks for letting us know this, very useful info. I have taken a published lentivirus (article: PMID= 16945906 ) and modified it to express a miRNA instead of siRNA. The order is Tetracycline promoter-GFP-miRNA-ubiquitin c promoter-rtRA3-IRES-Neo. The authors see GFP expression and siRNA expression from the 1 promoter. They use the Tet promoter and rtTA3 to make it conditional, but maybe you can just go CMVp-mCherry-pre-miR? I can see the GFP expression but haven't checked for the miRNA expression just yet.
Also, there are intronic miRNAs and the coding genes are correctly translated and expressed, so maybe a CMVp-pre-miR-mCherry would also work?