Functional assays plasmid encoding

An example of miR-dependent deadenylation of mRNA measured by this method is shown in Fig. 6.3C. In this case, HeLa cells (which express let-7) were transfected with a pDNA R-luc construct encoding three let-7 binding sites in the 3 UTR (3 X bulge), or with control constructs encoding either no sites (plasmid) or three mutated let-7 sites (3 x bulge mut) (constructs described in Pillai et al, 2005). Cells were harvested 24 h after transfection, RNA was purified for the PAT assay, and luciferase activity was measured from cell lysates. As reported previously, the presence of functional let-7 target sites results in specific repression of luciferase expression with very minor effects on mRNA stability (Pillai et al., 2005). The experiment in Fig. 6.3C demonstrates that the let-7 targeted reporter mRNA is selectively deadenylated. 

Efforts to test hypothesis two by biochemical reconstitution of a functional desaturase complex in vitro proved technically difficult and failed to provide a reproducible assay for functional expression of cloned lepidopteran desaturase sequences. In contrast, an in vivo expression system consisting of the yeast olel mutant and YEpOLEX plasmid confirmed hypothesis two and provided a technically facile and robust assay for determining the functional identities of many moth desaturase-encoding cDNAs. Particularly desirable features of this... 

To analyzes the functional elements in the 2.8 kb fragment, several deletions were made in the 5 and the 3 region in a plasmid containing TK promoter and the sequence encoding luciferase. Transfection assays were performed with these various deleted constructs in HeLa cells. Preliminary transfection results seem to suggest that this localized PPRE element is not the only one in controlling thiolase B expression by PPARa. 

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