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siRNA Expression Vectors with Selectable Markers For Long Term Studies in Mammalian Cells
Six new p
Silencer™ siRNA expression vectors are now available, each with an antibiotic resistance gene to facilitate selection in mammalian cultured cells. These new vectors are ideal for the long-term study of gene specific knockdown. Gene specific silencing in mammilian cells can be readily accomplished by inducing the RNA interference pathway with siRNAs. Advantages of using siRNA expression vectors over siRNAs prepared by chemical synthesis, in vitro transcription or RNase III digestion of long dsRNA include the ability to perform long term gene silencing studies and eliminating the need to work directly with RNA.
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 (See Why RNA Polymerase III Promoters? in sidebar). The insert that encodes this hairpin is designed to have two inverted repeats separated by a short spacer sequence (Figure 1). 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. (See Design of Vector Encoded siRNAs in sidebar). Once inside the cell, the vector constitutively expresses the hairpin RNA, which induces silencing of the target gene.
Figure 1. Schematic of a Typical siRNA Expression Vector. A. siRNA encoding insert. B. Vector. C. Hairpin siRNA.
Selectable Markers are Ideal for Long Term Studies
The use of mammalian siRNA expression vectors with antibiotic resistence genes provides 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. Transient use of an antibiotic can be used to enrich the population of cells that have taken up the marker-containing plasmid. This means that useful results can be obtained from experiments where transfer of the siRNA expression plasmid into the cells is inefficient.
In addition, use of selectable markers 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 several weeks.
In addition, use of selectable markers 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 several weeks.
siRNA Expression Vectors with Puromycin, Neomycin, and Hygromycin Selectable Markers
Six pSilencer siRNA Expression Vectors with antibiotic selectable markers are now available from Ambion (click here for comparison chart). These vectors feature the same 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 resistence gene.
All six siRNA expression plasmids allow for the selection of cells that have taken up the plasmid and are expressing the resistance gene.
To demonstrate the use of Ambion's new selectable marker containing pSilencer siRNA expression vectors, HeLa cells expressing GFP were transfected with pSilencer 2.1-U6 hygro encoding an siRNA to GFP and selected with the antibiotic hygromycin (Figure 2). Three weeks after transfection, GFP levels were analyzed by fluorescence microscopy. GFP levels were found to be reduced 94% as compared to cells transfected with pSilencer 2.1-U6 hygro without an insert. This reduction in gene expression was maintained for at least four weeks.
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.
Figure 2. Long Term Silencing of GFP with pSilencer™ 2.1-U6 hygro. HeLa cells expressing cycle 3 GFP were transfected with pSilencer 2.1-U6 hygro containing an insert encoding an siRNA targeting cycle 3 GFP under the control of the human U6 promoter (Panel A) or pSilencer 2.1-U6 hygro without an siRNA-encoding insert (Panel B). Following transfection, the cells were selected with hygromycin. Three weeks following selection, the cells were analyzed for GFP expression by fluorescence microscopy. Green: GFP. Blue: DAPI stained nuclei. GFP levels were remarkably reduced (94%) in cells transfected with the GFP siRNA-encoding pSilencer 2.1-U6 hygro siRNA Expression Vector as compared to those transfected with an "empty" siRNA expression vector.
- pSilencer 2.1-U6 puro and pSilencer 3.1-H1 puro contain the resistance gene pac, which confers resistance to puromycin.
- pSilencer 2.1-U6 hygro and pSilencer 3.1-H1 hygro contain the resistance gene hph, which inactivates hygromycin.
- pSilencer 2.1-U6 neo and pSilencer 3.1-H1 neo contain the neomycin resistance gene, which confers resistance to the antibiotic G418.
All six siRNA expression plasmids allow for the selection of cells that have taken up the plasmid and are expressing the resistance gene.
To demonstrate the use of Ambion's new selectable marker containing pSilencer siRNA expression vectors, HeLa cells expressing GFP were transfected with pSilencer 2.1-U6 hygro encoding an siRNA to GFP and selected with the antibiotic hygromycin (Figure 2). Three weeks after transfection, GFP levels were analyzed by fluorescence microscopy. GFP levels were found to be reduced 94% as compared to cells transfected with pSilencer 2.1-U6 hygro without an insert. This reduction in gene expression was maintained for at least four weeks.
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.
Figure 2. Long Term Silencing of GFP with pSilencer™ 2.1-U6 hygro. HeLa cells expressing cycle 3 GFP were transfected with pSilencer 2.1-U6 hygro containing an insert encoding an siRNA targeting cycle 3 GFP under the control of the human U6 promoter (Panel A) or pSilencer 2.1-U6 hygro without an siRNA-encoding insert (Panel B). Following transfection, the cells were selected with hygromycin. Three weeks following selection, the cells were analyzed for GFP expression by fluorescence microscopy. Green: GFP. Blue: DAPI stained nuclei. GFP levels were remarkably reduced (94%) in cells transfected with the GFP siRNA-encoding pSilencer 2.1-U6 hygro siRNA Expression Vector as compared to those transfected with an "empty" siRNA expression vector.
Why RNA Polymerase III 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 to 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 expression vectors with each of the three distinct promoters as the amount and location of siRNAs expressed from these promoters can vary differentially from cell type to cell type.
Design 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 precaution 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. Ambion recommends and uses inserts that encode a hairpin with a 19 nucleotide stem and a specific 9 base loop sequence (Figure 1 at left).
Tips on Using Selectable Marker Containing siRNA Expression PlasmidsAlthough antibiotic selectable markers permit the long term study of gene silencing effects, the actual length of time that a gene silencing experiment can be followed will depend on the identity and function of the target protein. Obviously, silencing of a gene essential for cell viability would be difficult to analyze over a long period. However, analysis of effects that do not appear for several days after reduction in the target mRNA levels would be good candidates for long term study using selectable marker containing siRNA expression vectors.
Before beginning a gene silencing experiment with a selectable marker containing siRNA expression vector, each cell line to be used should be tested for the minimum amount of antibiotic that can efficiently kill cells that do not contain the antibiotic resistence gene. The best way to do this is to generate a dose response curve with the antibiotic of choice. Because it is difficult to obtain stable clones from normal cells in culture using selectable marker containing plasmids, these plasmids should be primarily used in transformed and cancer cells.
Before beginning a gene silencing experiment with a selectable marker containing siRNA expression vector, each cell line to be used should be tested for the minimum amount of antibiotic that can efficiently kill cells that do not contain the antibiotic resistence gene. The best way to do this is to generate a dose response curve with the antibiotic of choice. Because it is difficult to obtain stable clones from normal cells in culture using selectable marker containing plasmids, these plasmids should be primarily used in transformed and cancer cells.
References
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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): 5515-20.
- 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: 948-58.
- 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: 500-5.
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