• COMPLETE KIT
SIMPLIFIES SHRNA
CLONING
• EFFECTIVE TARGET GENE
SUPPRESSION
• IDEAL FOR TRANSIENT
AND LONG-TERM GENE
SILENCING STUDIES
Simplify Creation of
shRNA Expression
Vector Constructs
As the use of RNA interference (RNAi)
techniques has expanded,
different ways to prepare RNA molecules have
emerged. An effective technique is a vector that
uses a pol III promoter to express a short hairpin
How shRNA Works
shRNA consisting of short inverted repeats separated by a small loop sequence is expressed from
the U6 pol III promoter (Figure 1). One inverted
repeat is complimentary to the gene target
sequence (sense). When expressed in mammalian
cells, the shRNA is processed into siRNA which
binds to the target sequence and initiates the
degradation of the target gene mRNA, thereby
suppressing its expression.
RNA (shRNA) to provide transient or long-term
target gene suppression.
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Stratagene now makes the preparation
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of shRNA constructs easy with the
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GeneEraser™ pGE-1 predigested cloning kit.
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Figure 1
Representation of the shRNA Expression Cassette
Transcribed RNA forms hairpin structure.
Effective Suppression
siRNA oligos are expensive to synthesize, and
are effective for only 5 to 7 days in suppressing
expression of your gene of interest. While
shRNA vectors are effective in creating longterm gene suppression, cloning direct repeat
sequences can be challenging.
108
STRATEGIES
16.4
The GeneEraser™ pGE-1 predigested cloning kit*
uses the pGE-1 vector for expression of shRNA
(Figure 2). The human U6 promoter has shown
the highest suppression of the luciferase control in
a wide variety of cell lines, consistently reducing
target gene expression by 80 to 95% (Figure 3).
The pGE-1 predigested vector has been digested
with BamH I and Xba I for directional insertion of
a shRNA insert.
Efficient Cloning
The GeneEraser pGE-1 predigested cloning kit
contains all you need to prepare your shRNA constructs, including the competent cells and annealing buffer for your DNA oligos. Simply anneal,
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Protein Function and Analysis
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Figure 2
The pGE-1 Vector
Figure 3
Efficient Suppression of Luciferase
Number of colonies
pGE-1 + test insert
pGE-1 only
1
1440
23
2
1232
30
3
1176
27
4
1240
20
5
1048
19
6
1144
23
Average
1213
24
CHO cells were co-transfected with 100 ng pCMV-Luc, 500 ng of pCMV-βgal, and
either 500 ng of pGE-1-neg Control Vector or 500 ng of pGE-1-shluc Control Vector
and assayed for luciferase expression.
M
1
2
3
4
5
6
M
% background = 2%
Table 1
High Number of Colonies with a Low Percentage
Background
ligate, and transform into the competent cells.
Using the kit results in a high number of colonies
with a low percentage background (Table 1 and
Figure 4). When a unique restriction enzyme site is
included in the hairpin loop, it is easy to determine
positive clones by restriction analysis. Only clones
containing the shRNA will linearize; empty clones
will migrate as supercoiled DNA (Figure 4). The test
insert included in the kit is a scrambled sequence
that contains a unique Hind III site that can also be
used as a negative control for experiments.
Control Kit Also Available
It can be difficult to set up new systems in your
lab, so we have provided the GeneEraser™ shRNA
expression control kit for researchers just starting
out in siRNA. This kit contains the vector pCMVluc that expresses luciferase**, pGE-1-shluc that
expresses a shRNA to suppress luciferase expression, and pGE-1-neg that expresses a scrambled
shRNA that does not suppress expression of any
known mammalian gene (Figure 3).
Figure 4
Easy Detection of Insert
Five different clones containing pGE-1 and shRNA test insert (lanes 1-5) and empty
vector (lane 6) were digested with Hind III and run on an agarose gel. The hairpin loop
in the shRNA test insert contains a unique Hind III restriction site. In this example, all of
the pGE-1 with test insert DNA were linearized by Hind III indicating the presence of the
shRNA test insert while the pGE-1 vector remained supercoiled.
GeneEraser™ pGE-1 Predigested Cloning Kit
pGE-1 predigested vector
shRNA negative test insert
10X annealing buffer
DNA ligase
10X ligase buffer
rATP
pGE-1-neg control vector
SCS-1 supercompetent cells
Quantity
Catalog
20 rxn
#240094
20 µg
20 µg
20 µg
500 µl
#240093
GeneEraser™ shRNA Expression Control Kit
pGE-1-shluc control vector
pGE-1-neg control vector
pCMV-luc control vector
SCS-1 host strain
* See license reference 2 on page 136
** See license reference 3 on page 136.
www.stratagene.com
STRATEGIES
16.4
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