This Ambion® vector is for the expression of siRNA. It has an antibiotic
resistance gene (puromycin) 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.
