Figure 1
Overview of GRIM system. Details are described in the Results and Methods sections. (A) GRIM.miRNA vectors are constructed by BP recombination between a linear miRNA donor and our GRIM destination vector. Each linear miRNA donor is created by extending 4 DNA primers. Common primers 1 and 2 are used to build every linear miRNA donor DNA, and contain identical AttB, LoxP, and FRT sequences, as shown. The middle portion of the linear miRNA donor is generated by DNA oligos containing miRNA sequences designed by the end-user or using our GRIM.REAPER software package (Additional File 4). BP recombination produces the GRIM.miRNAoff vector, which contains a LoxP-flanked 5' AttL1 site that inhibits miRNA production. The AttL1 site can be removed by Cre recombinase to produce the constitutively active GRIM.miRNAon vector. The entire U6 promoter (U6 prom) miRNA cassette is flanked by FRT sites, which can be utilized to shut down the system in the presence of Flp recombinase. (B) Predicted secondary structure of the LoxP-flanked AttL1 site, which inhibits miRNA production. Cre excision of AttL1 releases this inhibition, thereby permitting miRNA expression. The miLacZ sequence is shown here (blue indicates sense strand; red indicates antisense guide strand). Nucleotides in orange are stem and loop sequences derived from human miR-30a, except the 3' terminal poly U, which is an engineered termination signal for the pol III-dependent U6 promoter. The blue and yellow arrowheads indicate Drosha and Dicer cut sites, respectively.