siRNA Expression Vectors with Selectable Markers For Long Term Studies in Mammalian Cells
How siRNA Expression Vectors Work
Figure 1. Schematic of a Typical siRNA Expression Vector. A. siRNA encoding insert. B. Vector. C. Hairpin siRNA.
Selectable Markers are Ideal for Long Term Studies
In addition, use of selectable markers permits long term gene silencing studies of cells that take up the siRNA expression vector. Changes in phenotype due to reduced gene expression that may not be readily apparent only a few days after transfection can be followed over several weeks.
siRNA Expression Vectors with Puromycin, Neomycin, and Hygromycin Selectable Markers
- pSilencer 2.1-U6 puro and pSilencer 3.1-H1 puro contain the resistance gene pac, which confers resistance to puromycin.
- pSilencer 2.1-U6 hygro and pSilencer 3.1-H1 hygro contain the resistance gene hph, which inactivates hygromycin.
- pSilencer 2.1-U6 neo and pSilencer 3.1-H1 neo contain the neomycin resistance gene, which confers resistance to the antibiotic G418.
All six siRNA expression plasmids allow for the selection of cells that have taken up the plasmid and are expressing the resistance gene.
To demonstrate the use of Ambion's new selectable marker containing pSilencer siRNA expression vectors, HeLa cells expressing GFP were transfected with pSilencer 2.1-U6 hygro encoding an siRNA to GFP and selected with the antibiotic hygromycin (Figure 2). Three weeks after transfection, GFP levels were analyzed by fluorescence microscopy. GFP levels were found to be reduced 94% as compared to cells transfected with pSilencer 2.1-U6 hygro without an insert. This reduction in gene expression was maintained for at least four weeks.
The selectable marker-containing pSilencer vectors are supplied with (1) linearized and purified vector ready for ligation; (2) a DNA insert encoding a GFP-specific siRNA; (3) a circular, negative control pSilencer vector that expresses a scrambled control siRNA; and (4) 1X DNA Annealing Solution.
Figure 2. Long Term Silencing of GFP with pSilencer™ 2.1-U6 hygro. HeLa cells expressing cycle 3 GFP were transfected with pSilencer 2.1-U6 hygro containing an insert encoding an siRNA targeting cycle 3 GFP under the control of the human U6 promoter (Panel A) or pSilencer 2.1-U6 hygro without an siRNA-encoding insert (Panel B). Following transfection, the cells were selected with hygromycin. Three weeks following selection, the cells were analyzed for GFP expression by fluorescence microscopy. Green: GFP. Blue: DAPI stained nuclei. GFP levels were remarkably reduced (94%) in cells transfected with the GFP siRNA-encoding pSilencer 2.1-U6 hygro siRNA Expression Vector as compared to those transfected with an "empty" siRNA expression vector.
Why RNA Polymerase III Promoters?
Before beginning a gene silencing experiment with a selectable marker containing siRNA expression vector, each cell line to be used should be tested for the minimum amount of antibiotic that can efficiently kill cells that do not contain the antibiotic resistence gene. The best way to do this is to generate a dose response curve with the antibiotic of choice. Because it is difficult to obtain stable clones from normal cells in culture using selectable marker containing plasmids, these plasmids should be primarily used in transformed and cancer cells.
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