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.