We therefore investigated whether the higher expression levels of the exogenous gene after using weak promoters for selection was caused by increased T-DNA copy numbers.
The weaker ARG4 promoter used for selection marker cassettes enables selection at lower concentrations of Zeocin™ (i.e., 25 instead of 100 μg/ml) without obtaining false-positive clones.
The expression vectors included a Neo resistance gene driven by the SV40 promoter for clone selection.
To establish pancreatic beta cell lines, grafts were removed, dissociated and transduced with viral vectors expressing the neomycin resistance gene under the control of the insulin promoter for further selection of insulin-transcribing cells by culture in the presence of G418 (Fig. 3A).
Both of these fragments were inserted into a pPZP221 binary vector [ 53], which has a gentamicin selection marker driven by the 35 S promoter [ 54] for selection of transformed plants.
In addition to the GAL4-EGFP expression cassette, this plasmid contains the neomycin resistance gene under the control of the SV40 promoter and enhancer for selection of transfected cells in G418.
HT29 cells were then transfected with 500 ng of each siRNA plasmid and 100 ng of pcDNA G418 resistance promoter-less plasmid for selection of transfectants.
pBF24 is ϕBT1 int/attP-based integrating plasmid (10) harboring the eryAII gene downstream of the actIp promoter and containing ermE for selection (Fig. 2).
