Nucleofection of shRNA Vectors
In addition to or as an alternative to siRNA duplexes, shRNA vectors are frequently used for RNAi applications. Vectors expressing shRNA offer an advantage over siRNA duplexes in that they permit a longer-lived inhibition of target genes. As transfection efficiency is one of the two most important factors determining the effectiveness of RNAi, the ability of the Nucleofector technology to transfect a wide range of cells with high efficiencies significantly increases the range of cell types in which plasmid based RNAi systems can be used, such as primary neurons. This makes it is an ideal alternative to circumvent laborious viral transduction.
The optimal nucleofection conditions for a particular cell type are identical whether you are transfecting DNA or RNA. This makes it a simple matter to switch back and forth between substrates (for example, when testing the efficiency of new sequences as siRNA oligos prior to constructing siRNA-expressing vectors).
- Non-viral transfection (cost and time effective)
- Up to 90% transfection efficiency for DNA vectors even in suspension cell lines and primary cells
- 96-well Shuttle enabling shRNA library screening
96-well nucleofection of a non-viral shRNA vector into rat neurons
Quantitative down-regulation of target proteins using RNA interference.
Hippocampal neurons (E17) were transfected by nucleofection with the shRNA vector pSuperior targeting CDC10. A and B: Efficient nucleofection of pSuperior is shown by eGFP expression after 1 DIV (days in vitro). C and D: Western blot analysis (C) and quantification (D) of CDC10 down-regulation. In contrast to control transfections (siPum2, misPum2), CDC10 expression is significantly reduced in neuronal cultures transfected with pSuperior vectors targeting CDC10. E: Immunostaining of CDC10 (red fluorescence) shows reduced endogenous CDC10 protein levels in transfected neurons (green, T) after 4 DIV compared to untransfected cells (red, UT).
(Data kindly provided by M. Kiebler, Div. of Neural Cell Biology, Medical University of Vienna).
96-well nucleofection of a lentiviral shRNA vector into Jurkat cells
Knockdown of Fas surface expression on Jurkat cells by nucleofection of an anti-Fas shRNA vector.
Jurkat E6-1 (ATCC) were either nucleofected with 0.5 µg shRNA vector targeting Fas or 0.5 µg pmaxGFP as a negative control using the Cell Line 96-well Nucleofector Kit SE and program 96-CL-120. Cells were stained at various time points post nucleofection with an antibody directed against Fas and analyzed by flow cytometry. Staining intensity is normalized to negative control expression of Fas (set to 100% for each time point).
