This Ambion® vector is for the expression of siRNA. It has an antibiotic resistance
gene (neomycin) to enable selection of transfected cells and features the human RNA
polymerase III promoter (U6). Sufficient reagents are provided for 20 reactions.
• Select transfected cells to enrich the population of cells expressing your siRNA
• Eliminate the need to synthesize RNA oligonucleotides for RNAi experiments
• Supplied linearized and ready for ligation
Compensate for Low Transfection Efficiencies and Perform Long-Term Studies
The use of mammalian siRNA expression vectors with antibiotic selectable markers
conveys many benefits. Selectable markers can help compensate for poor plasmid
transfection efficiencies seen with some cell lines. In these cases, only a fraction
of the transfected cells express the siRNA, and reduction in target gene expression
with even a potent siRNA can be difficult to detect. Use of a selectable marker
and transient antibiotic selection permits only cells that have received the
marker-containing plasmid to live in the presence of antibiotic. Thus, all of these
cells should be exhibiting RNAi. Use of selectable markers also 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 a longer period of time.
How siRNA Expression Vectors Work
Vectors that express siRNAs within mammalian cells typically use an RNA polymerase III
promoter to drive expression of a short hairpin RNA that mimics the structure of an siRNA.
The insert that encodes this hairpin is designed to have two inverted repeats separated by
a short spacer sequence. One inverted repeat is complementary to the mRNA to which the
siRNA is targeted. A string of thymidines added to the 3' end serves as a pol III
transcription termination site. Once inside the cell, the vector constitutively expresses
the hairpin RNA. The hairpin RNA is processed into an siRNA, which induces RNAi of the
target gene.
Design of Vector Encoded siRNAs
In general, the selection of an siRNA target site for vectors is the same as that used for
designing siRNAs that will be introduced directly into cells, with the added caution that
strings of four or more thymidine or adenosine residues should be avoided to reduce the
possibility of premature termination of the transcript. The length of the inverted repeats
that encode the stem of the putative hairpin, the order of the inverted repeats, the
length and composition of the spacer sequence that encodes the loop of the hairpin, and the
presence or absence of 5' overhangs can vary within certain parameters. It is recommended
to use inserts that encode a hairpin with a 19-nucleotide stem and a specific 9-base loop
sequence.
