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.
Objective:
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).
Methodology:
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.
Results:
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…
