RiboBio can provide 3"UTR plasmid vector construction service based on pmiR-RB-Report (TM) target gene of human, mouse and rat genome. Among them hundreds of human genes have been constructed, please click the 3"UTR plasmid vector product page query of pmiR-RB-Report ™ target gene, the other genes have to be customized.
pmiR-RB-Report ™ Reporter Assay System is designed to detect whether miRNA and target gene exist a regulatory role. The reporting system can report fluorescence (hRluc,), whose downstream is constructed to 3"UTR region of target genes. We can verify whether miRNA regulate this target gene by monitoring the fluorescence; there is another correction fluorescence (hluc) used as stable internal reference for monitoring the efficiency of plasmid expression.
Figure 1. pmiR-RB-Report ™ plasmid map diagram.
1) Varying length of genes’ 3"UTR is generally considered to limit the construction length to no more than 2.5kb. The length of vectors with more than 2.5kb will be very difficult, the price and construction time should be consulted.
2) We propose to construct single point mutation vector so as to verify the effect of target point. The construction of mutant vector is based mainly on the stable binding region between the predicted miRNA and the 3"UTR of target genes(ie, seed area). The binding capacity of miRNA and the 3"UTR region of target genes is greatly reduced by more than one mutation, so as to determine the existence of miRNA regulation.
Figure 2. Binding sites of hsa-miR-520a-3p and the 3"UTR region of h-of AKT1.
Figure 3. The mutations are usually seed area mutations.