This project with the combination of coding and non-coding cassettes in one vector is actually the very first project on which the founders collaborated when they met.
The goal was to engineer a complex metabolic pathway, in that case the glycosylation machinery of a mouse cell lines, by simultaneously inducing the expression of one glycosyltransferase (FUT3) and decreasing the expression of another endogenous glycosyltransferase (B3GALT6).
The strategy chosen for this was to assemble combining coding and non-coding cassettes in one vector, that is the FUT3 ORF driven by a Pol II promoter, and a B3GALT6 specific shRNA cassette driven by a Pol III promoter. A selection cassette was also required to select stable transfectants of a previously engineered cell lines (based on murine 4T1 cell line), with limited option available (Hygromycin resistance imperatively).
FUT3 expression was expected catalyze the biosynthesis of a glycan ligand specific of the E-selectin, while B3GLAT6 expression was involved in the biosynthesis of another glycan recognized by P-selectin. In order to evaluate both individual effect of the cassettes, some vectors contained a FUT3 antisense ORF and/or a scramble shRNA as negative control of either.
Stable 4T1 transfectants were subjected to a flow cytometry analysis using the E or P selectin as probe to monitor the presence of each ligand on the cell surface. The analysis showed that sense FUT3 indeed increased E-selectin signal, while B3GALT6 shRNA decreased P-selectin one, accordingly to expectations, though with some variable efficiency depending on the construct.
Such strategy can be applied to several projects requiring both coding and non-coding sequences to be expressed simultaneously in a cellular model:
– Genome editing (CRISPR based)
– Long non-coding RNA (lncRNA) expressed together with a reporter genes
– Silencing assays in presence or absence of insensitive targets (nucleotidic variants of coding sequences).
– And so forth…