- Available with hygromycin, puromycin, and neomycin resistance markers
- Select transfected cells to enrich the population of cells expressing your siRNA
- Efficiently express siRNA hairpins with either the human U6 or H1 polymerase III promoter
- Eliminate the need to synthesize RNA oligonucleotides for RNAi experiments
Important Licensing Information
siRNA Expression Vectors with Selectable Markers
Ambion has made available pSilencer™ siRNA Expression Vectors with antibiotic selectable markers. These vectors feature the same human U6 and H1 RNA polymerase III promoters, ampicillin resistance gene, and E. coli origins of replication as the pSilencer 2.0-U6 and pSilencer 3.0-H1 siRNA Expression Vectors, but have the added benefit of an antibiotic selectable marker.
[For a chart comparing the attributes of all of our siRNA expression vectors, please click here.]
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. This means that useful results can be obtained from experiments that normally would be useless due to inefficient transfer of the siRNA expression plasmid into the cells.
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. For such long-term gene silencing studies Ambion recommends that pSilencer™ 4.1-CMV vectors should be used. The pSilencer 4.1-CMV vector features a modified CMV promoter that performs better than most pol III promoters under long-term selection. Using the pSilencer 4.1-CMV vector system scientists at Ambion have been able to produce cell lines that have reduced levels of GAPDH mRNA and protein for over 8 months.
Therefore, Ambion recommends that pSilencer 4.1-CMV vectors be used for long-term gene silencing studies. However, for both transient gene silencing studies and transient antibiotic selection of positively transfected cells, pSilencer 2.1 and 3.1 series of vectors could be used in addition to pSilencer 4.1-CMV vectors.
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.
About the Promoters
In most siRNA expression vectors described to date, one of three different RNA polymerase III (pol III) promoters is used to drive the expression of a small hairpin siRNA (1-5). These promoters include the well-characterized human and mouse U6 promoters and the human H1 promoter. RNA pol III was chosen to drive siRNA expression because it expresses relatively large amounts of small RNAs in mammalian cells and it terminates transcription upon incorporating a string of 3–6 uridines. The latter feature is especially important due to the apparent requirement that siRNAs have defined 3' termini that hybridize to an mRNA target (6). The U6 and H1 promoters used for siRNA vector design were selected because they are relatively simple and they lie completely upstream of the sequence being transcribed. This eliminates any need to include promoter sequence within the siRNA. Ambion provides antibiotic selectable marker containing siRNA expression vectors with your choice of either the human U6 or H1 promoter because the amount and location of siRNAs expressed from these promoters is expected to vary from cell type to cell type.
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 (1-5; see also the Instruction Manual and Guidelines for Designing Inserts for siRNA Expression Vector for details). Ambion recommends and uses inserts that encode a hairpin with a 19 nucleotide stem and a specific 9 base loop sequence.
Linearized pSilencer Vectors Are Supplied with Positive and Negative Controls
The selectable marker containing pSilencer vectors are supplied with (1) linearized and purified vector ready for ligation; (2) a DNA insert encoding a GFP-specific siRNA; (3) a circular, negative control pSilencer vector that expresses a scrambled control siRNA; and (4) 1X DNA Annealing Solution. All six siRNA expression plasmids allow for the selection of cells that have taken up the plasmid and are expressing the resistance gene.
pSilencer 2.1-U6 puro and pSilencer 3.1-H1 puro contain the resistance gene pac from Streptomyces alboniger, which encodes puromycin N-acetyl-transferase and conveys resistance to puromycin. pSilencer 2.1-U6 hygro and pSilencer 3.1-H1 hygro contain the resistance gene hph from Streptomyces hygroscopicus, which encodes a kinase that inactivates hygromycin. pSilencer 2.1-U6 neo and pSilencer 3.1-H1 neo contain the neomycin resistance gene from Tn5, which encodes an aminoglycoside 3'-phosphotransferase (3' APH II) and confers resistance to the antibiotic G418.
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For help identifying potential target sequences within your mRNA, use our siRNA Target Finder. Resulting target sequences can then be sent directly to our pSilencer Expression Vectors Insert Design Tool for the generation of the hairpin siRNA-encoding oligonucleotide inserts needed for use of these vectors. Or go straight to the Insert Design Tool and paste in your own siRNA target sequence. |
References
- Brummelkamp, TR, Bernards, R, and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550-553.
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Sui G, Soohoo C, Affar EB, Gay F, Shi Y, Forrester WC, and Shi Y. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 99(8): 5515-5520.
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Paddison, PJ, Caudy, AA, Bernstein, E, Hannon, GJ and Conklin DS. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Development 16:948-958.
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Lee NS, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, Rossi J. (2001) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 19: 500-505.
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Paul CP, Good PD, Winer I, Engelke DR. (2002) Effective Expression of Small Interfering RNA in human cells. Nat Biotechnol 19: 505-508.
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Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T. (2001) Functional anatomy of siRNA for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J 20: 6877-6888.
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