- Induce gene silencing in mammalian cultured cells
- CMV promoter, a polymerase (pol) II promoter, efficiently expresses siRNA in a broad range of cells
- Improved long-term silencing
- Compatible with more siRNA sequences
Important Licensing Information
Versatile Vectors for siRNA Expression
Ambion's pSilencer™ 4.1-CMV plasmid vectors are designed for gene silencing experiments in a broad range of cell lines. Unlike many commonly used vectors, the pSilencer 4.1-CMV vectors carry a modified RNA polymerase II-type CMV promoter (human cytomegalovirus immediate-early promoter) and an optimized SV40 polyadenylation signal to drive high-level siRNA expression. For stable transfections requiring long-term antibiotic selection, an SV40 promoter expresses one of three antibiotic resistance genes (hygromycin, neomycin, or puromycin). The pSilencer 4.1-CMV vector system is supplied with (1) Bam H1 and Hind III-linearized and purified vector ready for ligation; (2) a DNA insert encoding a GAPDH-specific siRNA; (3) a circular, negative control pSilencer vector that expresses a scrambled control siRNA; and (4) 1X DNA annealing solution.
For siRNA expression, the pol II-type CMV promoter has several advantages over pol III promoters such as U6 or H1. First, pol II will tolerate strings of 4 or more U’s within the siRNA sequence, unlike pol III which will terminate transcription after incorporation of a stretch of U’s. Second, the CMV promoter does not interfere with other transcription events (such as expression of the antibiotic resistance gene), making it easier to perform long-term gene silencing studies. Indeed, using the pSilencer 4.1-CMV vector system, Ambion scientists have been able to produce cell lines with reduced levels of GAPDH mRNA and protein lasting over 8 months.
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 (see siRNA Design Guidelines for details). 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.
Using pSilencer 4.1-CMV Vectors to Express microRNAs and mRNAs
Expression of hairpin siRNAs for gene silencing is only one application of the pSilencer 4.1-CMV vectors. These vectors can also be used to express microRNAs (miRNAs). Like siRNAs, miRNAs are expressed as longer hairpin molecules. These primary transcripts are then cleaved in the nucleus to 60-70 nt pre-miRNA hairpins. Pre-miRNAs are exported to the cytoplasm where they are processed into 21-24 nt miRNAs [3, 4]. Consequently, to efficiently express miRNA from a mammalian expression vector, 50-100 bases of extra sequence must be added to the flanking sides of the hairpin miRNA sequence. For more information about the design of inserts for miRNA expression, please contact an Ambion Technical Service Scientist. To view miRNA expression data, see Versatile Vectors for Expression of siRNA, miRNA, and mRNA.
In addition to providing high-level expression of mature siRNA and miRNA, the CMV promoter is excellent for stable or transient expression of protein in a wide range of mammalian cells. Ambion scientists have expressed luciferase and CAT reporter genes using pSilencer 4.1-CMV vectors. For more information, see Versatile Vectors for Expression of siRNA, miRNA, and mRNA.
References
- Xia H, Mao Q, Paulson H, Davidson B (2002) siRNA-mediated gene silencing in vitro and in vivo. Nature Biotechnology 20: 1006-10.
- Foecking MK, Hofstetter H (1986) Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene 145 (1): 101-5.
- Lee Y, et al., (2003) The nuclear RNaseIII Drosha initiates microRNA processing. Nature 425 (6956): 415-9.
- Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U (2004) Nuclear export of microRNA precursors. Science, 303 (5654): 95-8.
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