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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.
Accessory Products: For such long-term gene silencing studies Ambion® recommends that pSilencer™ 4.1-CMV vectors (SKU#s AM5777, AM5779, and AM5775) should be used. These vectors feature a modified CMV promoter that performs better than most pol III promoters under long-term selection.
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