A construct with fluorescent indicators for conditional expression of miRNA

Transgenic RNAi holds promise as a simple, low-cost, and fast method for reverse genetics in mammals. It may be particularly useful for producing animal models for hypomorphic gene function. Inducible RNAi that permits spatially and temporally controllable gene silencing in vivo will enhance the power of transgenic RNAi approach. Furthermore, because microRNA (miRNA) targeting specific genes can be expressed simultaneously with protein coding genes, incorporation of fluorescent marker proteins can simplify the screening and analysis of transgenic RNAi animals.

We sought to optimally express a miRNA simultaneously with a fluorescent marker. We compared two construct designs. One expressed a red fluorescent protein (RFP) and a miRNA placed in its 3' untranslated region (UTR). The other expressed the same RFP and miRNA, but the precursor miRNA (pre-miRNA) coding sequence was placed in an intron that was inserted into the 3'-UTR. We found that the two constructs expressed comparable levels of miRNA. However, the intron-containing construct expressed a significantly higher level of RFP than the intron-less construct. Further experiments indicate that the 3'-UTR intron enhances RFP expression by its intrinsic gene-expression-enhancing activity and by eliminating the inhibitory effect of the pre-miRNA on the expression of RFP. Based on these findings, we incorporated the intron-embedded pre-miRNA design into a conditional expression construct that employed the Cre-loxP system. This construct initially expressed EGFP gene, which was flanked by loxP sites. After exposure to Cre recombinase, the transgene stopped EGFP expression and began expression of RFP and a miRNA, which silenced the expression of specific cellular genes.

We have designed and tested a conditional miRNA-expression construct and showed that this construct expresses both the marker genes strongly and can silence the target gene efficiently upon Cre-mediated induction of the miRNA expression. This construct can be used to increase the efficiency of making cell lines or transgenic animals that stably express miRNA targeting specific genes.