Designing dual reporter system for studying microRNA functions in embryonic stem cells

Embryonic stem (ES) cells are cell populations isolated from the inner cell mass (ICM) of the blastocyst during early mammalian embryonic development. ES cell population is capable of undergoing infinite self-renewal in the presence of appropriate growth factors and differentiating into any specified cell lineage. MicroRNAs (miRNAs) are a large class of short (18-22 nucleotides), endogenously expressed RNA molecules that have been shown to be critical in many biological processes in metazoans, including mammalian embryo development. Conventional molecular biology experiments have been performed to gain insights about miRNA expression in ES cells. Our lab used real-time PCR methods to extensively study miRNA expression patterns in 13 different murine ES cell lines and their cognate embryoid bodies (EBs). Based on their expression profile, two sets of miRNAs were suggestive of their roles in self-renewal and differentiation.; To functionalize these interesting miRNAs, we plan to either suppress or enhance miRNA activities in ES cells with antisense oligonucleotides or synthetic pre-miRNAs, respectively. We designed a cellular miRNA sensing system consisting of a miRNA-regulated EYFP expressing transgene and an ECFP transgene as an internal control. ECFP and EYFP transgenes were stably transfected into NIH 3T3 cells sequentially. Attenuation in yellow fluorescence was observed by fluorescence microscopy in cells whose EYFP transgene contains miRNA interacting sequence. Fluorescence activated cell sorting (FACS) results revealed analogous yellow fluorescence patterns. Cyan fluorescence, however, was observed to be localized within endoplasmic reticulum (ER) and Golgi complex, indicating that auto-fluorescence, not cyan fluorescence from ECFP, was detected. FACS analysis with cells carrying only ECFP transgene revealed that the majority of these cells barely emit cyan fluorescence. These results demonstrate that inappropriate chromosomal integration or epigenetic modification of human CMV promoter could occur, resulting in low expression of ECFP. Control experiments with plain NIH 3T3 cells and cell lines carrying only EYFP transgene have to be accomplished to ensure that fluorescence detected at cyan and yellow channels is definitely from its corresponding protein without any interference. Furthermore, a more robust promoter has to replace human CMV promoter to drive the expression of ECFP to remove the inconsistencies from human CMV promoter.