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Drive Long-Term Silencing with CMV Vectors
Ambion's newest line of plasmid siRNA expression
vectors, pSilencer™ 4.1-CMV,
feature polymerase (pol) II promoters that drive transcription
of both an siRNA template and an antibiotic resistance gene.
The CMV promoter is used to drive siRNA expression. This strong
promoter is active in a broad range of cell types and performs
better than most pol III promoters under long term selection.
One use of plasmid-based siRNA expression
vectors is for long term gene silencing studies in mammalian
cells. Typically these vectors encode a hairpin siRNA under
the control of a polymerase III promoter and an antibiotic
resistance gene driven by a polymerase II promoter. While it
is possible to use such vectors to produce stable cell lines
expressing siRNAs that silence their targets, there are,
to date, very few published accounts of doing so (Xia 2002).
One explanation for the difficulty in producing such clones
is possible interference between the pol II and III promoters.
Although the exact mechanism is not understood, it is hypothesized
that when pol II and III promoters are in close proximity,
interference of transcription between the two promoters can
occur. This suggests that clones derived from a transfection/selection
experiment will either express the siRNA or the antibiotic
resistance gene but rarely express both. To overcome this potential
problem, we have constructed three pSilencer 4.1-CMV
siRNA Expression Vectors that express a hairpin siRNA as
well as an antibiotic resistance gene from pol II promoters.
The CMV promoter expresses the siRNA, and an SV40 promoter
expresses the antibiotic resistance gene. To demonstrate the
utility of these CMV promoter vectors, the vectors were engineered
to express an siRNA targeting GAPDH. The vectors were then
transfected into HeLa cells, which were in turn put under long
term antibiotic selection. This strategy produced cell lines
that have had reduced levels of GAPDH mRNA and protein for
over 8 months.
As shown in Figure 1, several pSilencer 4.1-CMV
transfected clones had reduced GAPDH protein and mRNA expression
compared to non-transfected HeLa cells. Panel B shows immunofluorescence
data demonstrating the relative fluorescence differences between
a clone with reduced levels of GAPDH versus a clone that did
not show significant GAPDH reduction.
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Figure 1. Long
Term Stable Reduction in GAPDH Levels. Both
mRNA expression levels and protein levels were analyzed
in these same clones using either real-time PCR or
immunofluorescence analysis. (A) Clones that
showed low levels of GAPDH protein expression also
showed low levels of GAPDH mRNA expression. (B) Representative
immunostaining data showing the relative fluorescence
differences between a clone having GAPDH reduced and
another clone that did not have a significant amount
of GAPDH knocked down. Immunofluorescence was performed
using gene specific primary antibodies and a fluorescein
conjugated secondary antibody and the resulting reduction
in protein expression was examined by fluorescence
microscopy. The cells were mounted with DAPI to stain
nuclei (Blue). Images were digitally captured and quantified
using Metamorph software.
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The pSilencer 4.1-CMV siRNA Expression
Vectors are provided with 1) linearized and purified vector
ready for ligation; 2) a DNA insert encoding a GAPDH-specific
siRNA; 3) a circular, negative control pSilencer vector
that expresses a scrambled control siRNA; and 4) 1X DNA Annealing
Solution.
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