We are developing a GFP construct to use as a marker for successful electroporation of a minigene into human dendritic cells. the purpose is to use the dendritic cells as potential targets for t cells to discover potential antigens in the mutated minigenes vs the wild type. the minigenes alone appear to be too short-- either for rna stability or translational stability or proccessing/presenting the gene as measured by a control. does anyone know the issue with electroporating IVT products for short minigenes? is it because the mRNA is unstable? it cannot get translated? or the short peptide product is not stable or processed? when we use tandem minigenes (~900 bp long) there is no issue- the peptide gets translated and we can detect reactivity to the mutated minigenes but when we do single gene electroporation, we do not get any reactivity with the same construct.
we want to couple the individual minigene with GFP as a reporter for the electroporation. should the minigene be at the N terminus or C terminus of the GFP marker? should we include a stop codon between? we do not need any functional peptide from the minigene, only need the peptide to get processed and presented to act as a target for cd4 and cd8 t cells. would a stop codon affect the p/p?
any help is appreciated!
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