How to overexpress miRs - (Sep/30/2010 )
I want to express a certain miR in some lymphoid cell lines and check if they target my gene of interest. What would be the best (more reliable and biological approximated) way to overexpress ectopically a microRNA? And the cheapest? Transfecting with an expressing vector or infecting with a lentiviral vector? Or just transfect with ds miR? And which promoter to choose? What is the most reliable expressing system... because there are a lot of companies now, what have worked for you?
I've been searching in this forum for this same question but the opinions diverge
Thank you for your help
There are several companies making miRNA expression vectors
Open Biosystem, System Biosciences, GeneCopoeia, and Biosettia make lentiviral miRNA expression vector
Oirgene makes plasmid-based miRNA expression vector.
System Biosciences and Origene use dual promoters to express microRNA and selection marker, the problem is you might not know whether the miRNA is expressing when you selection marker is expressed from the other promoter. GeneCopoeia uses H1 promoter for miRNA expression and I guess they only express the pre-miRNA. I wonder the processibility of mature miRNA in the absence of flanking sequences in its precusor. Open Biosystem use one promoter and miRNA precursor is cloned in the "exon". I don't understand how the RFP and puro-resistant gene can be expressed while the same mRNA is processed by Drosha. Biosettia's miRNA expression vector has miRNA precursor cloned in the EF1a intron and RFP-puro expressed from the same promoter. I think it's a smart way to overexpress miRNA.