A representative experimental output is shown in Figures 2 and 3 for targeting the expression of SECp43, which is involved in selenoprotein synthesis20,21, phosphoseryl-tRNA[Ser]Sec kinase (PSTK), which phosphorylates the seryl moiety on seryl-tRNA[Ser]Sec is an intermediate in selenocysteine synthesis22,23, and the selenoproteins, thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). In Figure 2, constructs encoding siSECp43 and siPSTK were stably transfected into NIH 3T3 cells and the expression of the corresponding mRNAs examined by northern blotting. Lane 1 shows that SECp43 has been effectively removed using a single siSECp43 targeting construct and lane 2 shows that PSTK was also effectively removed using a single siPSTK targeting construct. Lane 3 shows that SECp43 and PSTK were effectively and simultaneously removed using a siPSTK/siSECp43 double knockdown construct in which the two targeting regions are connected in tandem. In Figure 3, DT cells were stably transfected with a control construct, pU6, or with the siTR1/siGPx1 double knockdown construct and then transiently transfected with different expression vectors, the cells labeled with 75Se and the resulting labeled selenoproteins analyzed following electrophoresis of cell extracts. Lane 1 shows the expression of selenoproteins in cells stably transfected with the control vector and lane 2 shows that both TR1 and GPx1 were effectively removed by the double targeting vector. Re-introduction of the TR1 or GPx1 wild type genes did not result in expression of these proteins due to the presence of the corresponding siRNAs generated from the stably transfected vector (lanes 3 and 5), but these proteins were expressed in cells transiently transfected with the vectors carrying TR1 and/or GPx1 genes with mutations in the regions corresponding to the siRNAs in order to circumvent the targeting regions (lanes 4, 6, 7).
siRNA constructs for knockdown of SECp43<sup>21</sup> and/or PSTK<sup>22,23</sup>, and TR1 and/or GPx1<sup>16</sup> were generated as given in the references \(see also Figure 1). The sequences of SECp43 \(nucleotides 594-612), PSTK \(467-494), TR1 \(1993-2014) and GPx1 \(803-821) were selected as knockdown targeting regions. To replace TR1 and GPx1, mouse TR1 and GPx1 genes were cloned into pcDNA3.1<sup>16</sup>. Mutations in the siRNA target region were introduced by PCR using mutant primers \(TR1 sense 5’-gtcttagtctca **aggtaccta** tgtctaatgtc-3’ and GPx1 sense 5’- **gc** ga **g** ag **a** tgg **g** ttca **a** ta-3’ wherein the bolded letters indicate mutated nucleotides) that were designed to circumvent the corresponding siRNAs.
Development of a construct encoding multiple siRNAs targeting genes for their simultaneous removal expands the usefulness of the already established powerful tool of RNAi technology. It provides a simple, rapid and effective means of generating stably transfected siRNA cell lines. Furthermore, this approach permits us to target a variety of genes to elucidate their possible interplay and/or their loss on cell function and broadens our approaches in therapeutic strategies. The fact that we can also replace gene expression either individually or collectively provides an alternative means of assessing the interplay of different proteins as well as their individual or collective effect on overall cellular function.