Chapter 2: RNA Interference

RNA Interference

Introduction

In this chapter we provide a brief overview of the RNA interference (RNAi) process and discuss technologies and products that can be used for RNAi experiments. A protocol is provided for the enzymatic synthesis of double-stranded RNA in vitro that provides an inexpensive alternative to chemical synthesis of RNAs. We also discuss design and selection of short interfering RNA (siRNA) sequences and describe a vector system, the psiCHECK™ Vectors, that can be used to screen potential siRNA target sequences for effectiveness during RNAi optimization. In addition, two DNA-directed RNAi (ddRNAi) systems specifically designed for expression of small hairpin RNAs in mammalian cells are described. These are: 1) the siLentGene™-2 U6 Hairpin Cloning Systems, which provide a quick, PCR-based method for testing short hairpin RNA (shRNA) target sequences and a selection of vectors for ligation of these targets; and 2) the siSTRIKE™ U6 Hairpin Cloning Systems, which provide a cloning-based approach to allow fast, easy ligation and expression of hairpin oligonucleotides. Various strategies for delivery of siRNA to target cells are discussed, and example protocols for transient and stable tranfection of mammalian cells are provided. Finally, methods for quantitating target gene suppression are briefly summarized.

Overview and Mechanism of RNAi

RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) suppresses expression of a target protein by stimulating the specific degradation of the target mRNA (for reviews see Hannon, 2003; Caplen, 2004; Fuchs et al. 2004; Betz, 2003a). RNAi has been used to study loss of function of a variety of genes in several organisms including, various plants, Caenorhabditis elegans and Drosophila, and permits loss-of-function genetic screens and rapid tests for genetic interactions in mammalian cells (Hannon, 2002; Williams et al. 2003).

RNAi involves a multistep process (Figure 2.1). dsRNA is recognized by an RNase III family member (e.g., Dicer in Drosophila) and cleaved into siRNAs of 21–23 nucleotides (Agrawal et al. 2003; Elbashir et al. 2001b; Bernstein et al. 2001; Hammond et al. 2000). These siRNAs are incorporated into an RNAi targeting complex known as RISC (RNA-induced silencing complex), which destroys mRNAs homologous to the integral siRNA (Hammond et al. 2000; Bernstein et al. 2001). The target mRNA is cleaved in the center of the region complementary to the siRNA (Elbashir et al. 2001c), with the net result being rapid degradation of the target mRNA and decreased protein expression.

RNAi has revolutionized the study of gene function, and is being explored as a therapeutic tool (for reviews, see Dorsett and Tuschl, 2004; Hannon and Rossi, 2004). For example, RNAi has been used to identify gene products essential for cell growth (Harborth et al. 2001), to cause subtype and species-specific knockdown of various protein kinase C (PKC) isoforms in both human and rat cells (Irie et al. 2002), and to specifically target degradation of an oncogene product (Wilda et al. 2002). RNAi has also been used to specifically target and prevent viral infections by HIV-1 and HCV in cell culture (Park et al. 2002) and intact animals (McCaffrey et al. 2002). These observations open the field for further studies toward novel gene therapy approaches for anti-cancer or anti-viral treatments using siRNAs or shRNAs.

An animated presentation illustrating the entire RNAi process is available on the Nature web site.

RNAi as a Tool for Targeted Inhibition of Gene Expression

The use of long dsRNAs (>400bp) has been successful in generating RNA interference effects in many organisms including Drosophila (Misquitta and Paterson, 1999), zebrafish (Wargelius et al. 1999), Planaria (Sanchez-Alvarado et al. 1999) and numerous plants (Jorgensen, 1990, Fukusaki et al. 2004, Jensen et al. 2004). In mammalian systems, siRNA molecules of 21–22 nucleotides or short hairpin RNAs (shRNAs) are used to avoid endogenous nonspecific antiviral responses that target longer dsRNAs (Caplen et al. 2001, Elbashir et al. 2001a). Yu et al. (2002) and others (Brummelkamp et al. 2002b; McManus et al. 2002; Sui et al. 2002; Xia et al. 2002; Barton and Medzhitov, 2002) demonstrated that shRNAs bearing a fold-back, stem-loop structure of approximately 19 perfectly matched nucleotides connected by various spacer regions and ending in a 2-nucleotide 3′-overhang can be as efficient as siRNAs at inducing RNA interference. si/shRNAs can induce specific gene silencing in a wide range of mammalian cell lines without leading to global inhibition of mRNA translation (Caplen et al. 2001; Elbashir et al. 2001a; Paddison et al. 2002).

Generation of Short Interfering RNAs

siRNAs are the main effectors of the RNAi process. These molecules can be synthesized chemically or enzymatically in vitro (Micura, 2002; Betz, 2003b; Paddison et al. 2002) or endogenously expressed inside the cells in the form of shRNAs (Yu et al. 2002; McManus et al. 2002). Plasmid-based expression systems using RNA polymerase III U6 or H1, or RNA polymerase II U1, small nuclear RNA promoters, have been used for endogenous expression of shRNAs (Brummelkamp et al. 2002b; Sui et al. 2002, Novarino et al. 2004).

Rational Design of Effective siRNA Probes

Design of the siRNA sequence is crucial for effective gene silencing. Rational design strategies for effective siRNAs are being developed based on an understanding of RNAi biochemistry and of naturally occurring microRNA (miRNA) function. Several groups have proposed basic empirical guidelines for designing effective siRNAs that can be applied to the selection of potential target sequences (Chiu and Rana, 2002; Khvorova et al. 2003; Schwarz et al. 2003; Hseih et al. 2004; Reynolds et al. 2004; Ui-Tei et al. 2004). In addition, strategies for experimentally screening effective siRNAs from pools of potential siRNAs are being developed (Kumar et al. 2003; Vidugiriene et al. 2004a) and will remain a useful tool until potent siRNAs can be predicted accurately for each target gene.

Delivery of siRNA

The efficient delivery of siRNAs is a vital step in RNAi-based gene silencing experiments. Synthetic siRNAs can be delivered by electroporation or by using lipophilic agents (McManus et al. 2002; Kishida et al. 2004). siRNAs have been used successfully to silence target genes, however, these approaches are limited by the transient nature of the response. The use of plasmid systems to express small hairpin RNAs helps overcomes this limitation by allowing stable suppression of target genes (Dykxhoorn et al. 2003). Various viral delivery systems have also been developed to deliver shRNA-expressing cassettes into cells that are difficult to transfect, creating new possibilities for RNAi usage (Brummelkamp et al. 2002a; Rubinson et al. 2003). Successful delivery of siRNAs in live animals has also been reported (Hasuwa et al. 2002; Carmell et al. 2003; Kobayashi et al. 2004).

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siRNA Design and Optimization

Design of Target Sequences

Identifying an optimal target sequence is critical to the success of RNA interference experiments. Since it is not possible to predict the optimal siRNA sequence for a given target, multiple siRNAs will usually need to be evaluated. Recommendations for the design of siRNAs are constantly being improved upon as knowledge of the RNAi process continues to expand. At the time of writing this chapter, the recommendations are as follows: siRNA target sequences should be specific to the gene of interest and have ~20–50% GC content (Henshel et al. 2004). Ui-Tei et al. (2004) report that siRNAs satisfying the following conditions are capable of effective gene silencing in mammalian cells 1) G/C at the 5′ end of the sense strand; 2) A/U at the 5′ end of the antisense strand; 3) at least 5 A/U residues in the first 7 bases of the 5′ terminal of the antisense strand; 4) no runs of more than 9 G/C residues. Additionally, primer design rules specific to the RNA polymerase used will apply. For example, for RNA polymerase III, the polymerase that transcribes from the U6 promoter, the preferred target sequence is 5′-GN₁₈-3′. Runs of 4 or more Ts (or As on the other strand) serve as terminator sequences for RNA polymerase III and should be avoided. In addition, regions with a run of any single base should be avoided (Czauderna et al. 2003). It is generally recommended that the mRNA target site be at least 50–200 bases downstream of the start codon (Sui et al. 2002; Elbashir et al. 2002, Duxbury and Whang, 2004) to avoid regions in which regulatory proteins might bind.

Several online design tools are available to assist in identifying potential siRNA targets. The siRNA Designer provides such a tool for identifying target sequences for use with Promega RNA interference systems. This tool searches for sequences satisfying siRNA design recommendations and also incorporates a BLAST search capability to ensure that selected sequences are specific to the gene of interest, an important requirement to ensure specificity and minimize off-target effects. The siRNA Designer program designs oligonucleotides for use with the siSTRIKE™ U6 Hairpin Cloning Systems (Cat.# C7890, C7900, C7910, C7920), the siLentGene™-2 U6 Hairpin Cloning Systems (Cat.# C7860, C8060, C8070, C8080) and the T7 RiboMAX™ Express RNAi System (Cat.# P1700). The siRNA Designer analyzes input DNA or RNA sequences for regions that fit siRNA design requirements and displays siRNAs that could target these regions, along with the sequences of the oligonucleotides needed to produce these siRNAs with the chosen system.

Use of Reporter Genes for RNAi Optimization

Not all siRNAs directed against a target gene are equally effective in suppressing expression of that target in mammalian cells. Therefore, it is important to identify siRNA sequences that are effective inhibitors of target gene expression. Although rational designs for selection of potential target sequences have been encouraging in generating effective siRNAs, accurate prediction of the most effective siRNAs still remains to be achieved. Current screening technologies are based on semi-quantitative, time-consuming methods and are not easily modified to perform rapid, simultaneous screening of multiple siRNA/shRNA sequences. However, as the field of RNAi advances, and more high-throughput applications are adopted, there is a growing need for rapid, quantitative screening to confirm siRNA effectiveness (Kumar et al. 2003; Mousses et al. 2003).

Recently, several quantifiable procedures that use reporter genes to help rapidly identify effective siRNAs have been developed. In these approaches, the change in expression of a reporter gene fused to a target gene is used as an indicator of the effectiveness of an RNAi methodology. Here, we describe the psiCHECK™ Vector system, which is based on use of the bioluminescent Renilla luciferase reporter gene. The psiCHECK™ Vectors offer several advantages compared to other fusion approaches such as green fluorescent protein (GFP)- or Flag-tag-based methods. Measurement of net fluorescence from GFP in cell culture can be difficult and, in most cases, a flow cytometer is required for quantitation. Flag-tag quantitation requires Western blot analysis, which can be time-consuming. The high sensitivity of bioluminescence detection can readily tolerate lower expression levels, and introduction of a second reporter gene, firefly luciferase, allows normalization of changes in Renilla luciferase expression, making the psiCHECK™ Vector approach more robust and giving greater reproducibility of results.

The psiCHECK™-1 and -2 Vectors allow quantitative selection of optimal siRNA target sites and can be adapted for use in high-throughput applications. Figure 2.2 provides a basic illustration of how the psiCHECK™ Vectors are used. Both vectors contain a synthetic version of the Renilla luciferase (hRluc) reporter gene for monitoring RNAi activity. Several restriction sites are included 3′ of the luciferase translational stop codon, allowing creation of transcriptional fusions between the gene of interest and the Renilla luciferase reporter gene. Because of the presence of a stop codon in-frame with the Renilla luciferase open reading frame, no fusion protein is produced. Consequently, there is no need to maintain frames when inserting the target gene. Also, toxic genes or gene fragments can be analyzed using this design without the danger of these genes killing the transfected cells.

The psiCHECK™-1 Vector (Cat.# C8011) is recommended for monitoring RNAi effects in live cells. Changes in Renilla luciferase activity can be measured with the EnduRen™ Live Cell Substrate (Cat.# E6481). This approach permits continuous monitoring of intracellular luminescence. Renilla luciferase expression can be monitored continuously for 2 days without interfering with normal cell physiology.

The psiCHECK™-2 Vector (Cat.# C8021) contains an additional reporter gene, a synthetic firefly luciferase gene (hluc+), and is designed for endpoint lytic assays. Inclusion of the firefly luciferase gene permits normalization of changes in Renilla luciferase expression to firefly luciferase expression. Renilla and firefly luciferase activities can be measured using either the Dual-Luciferase^® Reporter Assay System (Cat.# E1910) or the Dual-Glo™ Luciferase Assay System (Cat.# E2920).

To use the psiCHECK™ Vectors for screening siRNA targets, the gene of interest is cloned into the multiple cloning region located 3´ to the synthetic Renilla luciferase gene and its translational stop codon. After cloning, the vector is transfected into a mammalian cell line, and a fusion of the Renilla gene and the target gene is transcribed. Functional Renilla luciferase is translated from the intact transcript. Depending on your experimental design, vectors expressing shRNA or synthetic siRNA can be either co-transfected simultaneously or sequentially. If a specific shRNA/siRNA effectively initiates the RNAi process on the target RNA, the fused Renilla target gene mRNA sequence will be degraded, resulting in reduced Renilla luciferase activity. An example protocol and experimental results illustrating the use of the psiCHECK™-2 Vector to evaluate multiple potential shRNAs are given below and in Figure 2.3, respectively.

Example Protocol for shRNA Target Screening Using the psiCHECK™-2 Vector

This protocol describes a co-transfection experiment using the psiCHECK™-2 Vector to screen a set of shRNAs potentially targeting p53 mRNA. In this experiment, each shRNA target was cloned into the psiLentGene™ Basic Vector (Section IV.A) and transfection into HEK-293T cells was performed using the TransFast™ Transfection Reagent (Cat.# E2431). This protocol is provided as an example. Conditions for each individual experimental system will require optimization.

Materials Required:

• psiCHECK™-2 Vector (Cat.# C8021) containing target gene
• psiLentGene™-2 Basic Vector (Cat.# C7860) containing shRNA sequence(s)
• TransFast™ Transfection Reagent (Cat.# E2431)
• cultured cells, serum-free and complete media
• Dual-Luciferase^® Reporter Assay System (Cat.# E1910)
• luminometer

1. One day before transfection, plate 3 x 10³ cells in 100µl/well (in a 96-well plate) in complete growth medium without antibiotics.

2. Add serum-free medium (35µl per assay well) to a sterile, 1.5ml tube. Add 0.02–0.08µg of the psiLentGene™ Basic Vector expressing shRNA and 0.02–0.04µg of the psiCHECK™-2:p53 vector for each well of the assay. Mix by gentle pipetting.

3. Add 0.3µl/well Transfast™ Reagent dropwise to the tube containing serum-free medium and DNA prepared in Step 2. Vortex thoroughly. Incubate at room temperature for 10–15 minutes.

4. In addition to testing various shRNA targets, positive and negative control constructs should be included. In this example, five psiLentGene™ constructs containing shRNAs directed against the p53 target gene, psiLentGene™ constructs containing shRNA directed against Renilla luciferase (positive control), and psiLentGene™ constructs containing a nonspecific shRNA (negative control) were tested.

5. Remove the medium from the cells in the 96-well plate and add 35µl of the DNA/transfection reagent mixture to each well

6. Incubate at 37°C for 1 hour.

7. Add 100µl of complete growth medium to each well and incubate for a further 24–48 hours.

8. Measure firefly and Renilla luciferase activities using the Dual-Luciferase^® Reporter Assay System.

9. Determine the Renilla/firefly luciferase activity ratio for each well. To calculate suppression levels, compare the Renilla/firefly ratio in cells transfected with each test shRNA to that in cells transfected with control shRNA.

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Enzymatic Synthesis of RNA in Vitro

siRNA synthesis in vitro provides a useful alternative to the potentially expensive chemical synthesis of RNA (Figure 2.4). The method relies on T7 phage RNA polymerase to produce individual sense and antisense strands that are annealed in vitro prior to delivery into the cells of choice (Fire et al. 1998; Donze and Picard, 2002; Yu et al. 2002, Shim et al. 2002).

The T7 RiboMAX™ Express RNAi System (Cat.# P1700) is an in vitro transcription system designed for rapid production of milligram amounts of double-stranded RNA (dsRNA). The system can be used to synthesize siRNAs for use in mammalian systems (Figure 2.5; Betz, 2003b, Hwang et al. 2004) or longer interfering RNAs for nonmammalian systems (Betz and Worzella, 2003; Betz, 2003c). The DNA templates for in vitro transcription of siRNAs are a pair of short, duplex oligonucleotides that contain T7 RNA polymerase promoters upstream of the sense and antisense RNA sequences. Each oligonucleotide of the duplex is a separate template for the synthesis of one strand of the siRNA. The separate short RNA strands that are synthesized are then annealed to form siRNA.

In Vitro Synthesis of dsRNA for Use in Nonmammalian Systems

RNAi experiments in nonmammalian systems are typically performed with dsRNA of 400bp or larger (Elbashir et al. 2001b; Yang et al. 2000, Hammond et al. 2000). The minimum size of dsRNA recommended for RNAi in these systems is ~200bp. In general, templates for transcription of dsRNA for use in RNAi experiments correspond to most or all of the target message sequence. Data suggests that longer dsRNA molecules are more effective on a molar basis at silencing protein expression, but higher concentrations of smaller dsRNA molecules may have similar silencing effects. Data generated at Promega suggests that smaller dsRNAs can be as effective and efficient at inducing RNAi in nonmammalian systems (Betz, 2003c).

In the T7 RiboMAX™ Express RNAi System, dsRNA production requires a T7 RNA polymerase promoter at the 5′-ends of both DNA target sequence strands. to achieve this, separate DNA templates, each containing the target sequence in a different orientation relative to the T7 promoter, are transcribed in two separate reactions. The resulting transcripts are mixed and annealed post-transcriptionally. DNA templates can be created by PCR or by using two linearized plasmid templates, each containing the T7 polymerase promoter at a different end of the target sequence.

See the T7 RiboMAX™ Express RNAi System Technical Bulletin #TB316 for a protocol for in vitro synthesis of dsRNA for RNAi in nonmammalian systems.

In Vitro Synthesis of siRNA for Use in Mammalian Systems

Figure 2.6 outlines the protocol for synthesis of siRNA using the T7 RiboMAX™ Express RNAi System. The initial step is generating the DNA template, which consists of two DNA oligonucleotides annealed to form a duplex. Generally 20pmol of duplex oligonucleotides are required per 20µl in vitro transcription reaction. Using the RiboMAX™ Express T7 Buffer and Enzyme Mix allows efficient synthesis of RNA in as little as 30 minutes. The annealed DNA oligonucleotide template is removed by a DNase digestion step, and the separate small RNA strands (sense and antisense) are annealed to form siRNA. The siRNA is precipitated using sodium acetate and isopropanol, and the resuspended product can be analyzed on polyacrylamide gels for size and integrity. Quantitation of the siRNA can be accomplished by either gel analysis or RiboGreen^® analysis (Molecular Probes).

Materials Required:

• T7 RiboMAX™ Express RNAi System (Cat.# P1700)
• 2X oligo annealing buffer (20mM Tris-HCl [pH 7.5], 100mM NaCl)
• nuclease-free water
• gene-specific oligonucleotides
• isopropanol
• 70% ethanol

Designing DNA Oligonucleotides

The target mRNA sequence selected must be screened for the sequence 5′-GN₁₇C-3′. The generation of 3–5 different siRNA sequences for a particular target is recommended to allow screening for the optimal target site. The oligonucleotides consist of the target sequence plus the T7 RNA polymerase promoter sequence and 6 extra nucleotides upstream of the minimal promoter sequence to allow for efficient T7 RNA polymerase binding. Details on design of oligonucleotides for use with this system are found in the T7 RiboMAX™ Express RNAi System Technical Bulletin #TB316.

For further assistance, an siRNA finder and oligo design tool that specifically selects siRNA targets for use with the RiboMAX™ System is available in the siRNA Designer. Simply select the "T7 RiboMAX™ Express RNAi System: siRNA" option.

Annealing DNA Oligonucleotides

1. Resuspend DNA oligonucleotides in nuclease-free water to a final concentration of 100pmol/µl.

2. Combine each pair of DNA oligonucleotides to generate either the sense strand RNA or antisense strand RNA templates as follows:

3. Heat at 90–95°C for 3–5 minutes, then allow the mixture to cool slowly to room temperature. The final concentration of annealed oligonucleotide is 10pmol/µl. Store annealed oligonucleotide DNA template at either 4°C or –20°C.

Synthesizing Large Quantities of siRNA

1. Set up the reaction at room temperature. The 20µl reaction may be scaled as necessary (up to 500µl total volume; use multiple tubes for reaction volumes >500µl). Add the components in the order shown below. For each siRNA, two separate reactions must be assembled as each RNA strand is synthesized separately, and then mixed following transcription.

2. Incubate for 30 minutes at 37°C.

Removing the DNA Template and Annealing siRNA

The DNA template can be removed by digestion with DNase following the transcription reaction. RQ1 RNase-Free DNase (Cat.# M6101) has been tested for its ability to degrade DNA while maintaining the integrity of RNA. If accurate RNA concentration determination is desired, the RNA should be DNase-treated and purified to remove potentially inhibitory or interfering components.

1. To each 20µl transcription reaction, add 1µl RQ1 RNase-Free DNase and incubate for 30 minutes at 37°C.

2. Combine separate sense and antisense reactions and incubate for 10 minutes at 70°C, then allow the tubes to cool to room temperature (approximately 20 minutes). This step anneals the separate short sense and antisense RNA strands, generating siRNA.

Purifying siRNA

1. Add 0.1 volume of 3M Sodium Acetate (pH 5.2) and 1 volume of isopropanol. Mix and place on ice for 5 minutes. The reaction will appear cloudy. Spin at top speed in a microcentrifuge for 10 minutes.

2. Carefully aspirate the supernatant, and wash the pellet with 0.5ml of cold 70% ethanol, removing all ethanol following the wash. Air-dry the pellet for 15 minutes at room temperature, and resuspend the RNA sample in nuclease-free water in a volume 2–5 times the original reaction volume (at least 2 volumes are required for adequate resuspension). Store at –20°C or –70°C.

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DNA-Directed RNAi

DNA-directed RNA interference (ddRNAi) involves the use of DNA templates to synthesize si/shRNA in vivo. ddRNAi relies on U6 or H1 [RNA polymerase III], or U1 [RNA polymerase II]) promoters for the expression of siRNA target sequences that have been transfected into mammalian cells (Miyagishi and Taira, 2002; Brummelkamp et al. 2002b; Novarino et al. 2004). si/shRNA target sequences can be generated by PCR, creating “expression cassettes” that can be transfected directly into cells (Csiszar et al. 2004; Castanotto et al. 2002) or cloned into expression vectors (Sui et al. 2002; Paul et al. 2002; Gou et al. 2003; Yu et al. 2002). PCR generation is recommended when rapid screening of numerous siRNAs is desired. Cloning-based approaches that allow direct ligation of hairpin oligonucleotides into a ddRNAi vector provide another method for quickly and easily screening various targets (Bailey et al. 2004; Vidurigiene et al. 2004b; Vidurigiene et al. 2004c). Screening can also be performed using synthetic RNAs, but this can become expensive for numerous targets.

Vector-based approaches also offer the potential of stable, long-term inhibition of gene expression by providing siRNAs on plasmids that allow selection of transfected cells. Vectors with markers such as puromycin, neomycin or hygromycin can be used for suppression of target genes for several weeks or longer. Transfection with synthetic siRNAs allows for only a transient measurement (usually 48–72 hours) of the RNAi effect.

The success of ddRNAi depends on several parameters including generation of vectors containing full-length sh/siRNA sequences, delivery of those vectors into cells, and expression of the si/shRNA constructs. Most strategies for cloning siRNA target sequences into expression vectors utilize the design of a hairpin structure. This design consists of two inverted repeats separated by a short spacer sequence (loop sequence). After transcription by RNA polymerase, the inverted repeats anneal and form a hairpin, which is then cleaved by Dicer to form an siRNA.

The siLentGene™-2 U6 Hairpin Cloning Systems and the siSTRIKE™ U6 Hairpin Cloning Systems are two systems that facilitate easy expression of shRNAs in vivo by ddRNAi-based methods. The siLentGene™ Systems provide the ability to directly transfect PCR-generated cassettes into cells for rapid screening of multiple shRNA targets. The siSTRIKE™ Systems provide a simple, cloning-based approach, allowing ligation of potential shRNA target sequences into vectors that allow transient or stable expression in mammalian cells. Overviews of both the siLentGene™ and siSTRIKE™ Systems are provided in Sections IV.A and IV.B, below.

siLentGene™-2 U6 Hairpin Cloning Systems

The siLentGene™-2 U6 Hairpin Cloning Systems (Cat.# C7860, C8060, C8070, C8080) are used primarily for screening siRNA targets that may then be cloned into a vector (Vidugiriene et al. 2004b). An overview of the siLentGene™-2 U6 Hairpin Cloning System procedure is shown in Figure 2.7. A DNA cassette containing a U6 promoter, a hairpin siRNA target sequence and the transcription termination sequence is generated by a single PCR amplification. The resulting PCR product can be directly transfected into human cells for rapid screening of optimal target sequences.

Once optimal target sequences are determined, the desired PCR products generated using the siLentGene™-2 U6 Hairpin Cloning Systems can be subcloned into plasmid vectors containing markers for selection of stable transfectants (Figure 2.8). The vectors are predigested, dephosphorylated and ready to use for direct subcloning of PCR products, and they also provide blue/white selection, allowing easy identification of recombinants on indicator plates. The psiLentGene™ Vectors contain the Amp^r gene, which confers resistance to ampicillin and allows selection in E. coli. The psiLentGene™ Vectors are also designed so the successful ligation of full-length PCR inserts regenerates an EcoR V site, providing a convenient method to confirm the presence of the desired insert.

siSTRIKE™ U6 Hairpin Cloning Systems

When an optimized RNAi target sequence is known, or only a few sequences are being evaluated, the siSTRIKE™ U6 Hairpin Cloning Systems (Cat.# C7890, C7900, C7910, C7920) should be used. In the siSTRIKE™ Systems, two short DNA oligonucleotides are synthesized and annealed to form a DNA insert that contains the hairpin siRNA target sequence. Upon annealing, the oligonucleotide forms ends that are compatible with the ends of the linearized psiSTRIKE™ Vector and thus facilitate sticky-end ligation (Figure 2.9). The linearized plasmids supplied with the system contain the human U6 promoter. Once transfected, RNA polymerase III transcribes the hairpin target sequences to generate hairpin shRNAs in vivo.

All the psiSTRIKE™ Vectors contain the Amp^r gene, which confers resistance to ampicillin and allows selection in E. coli. Three of the five psiSTRIKE™ Vectors: psiSTRIKE™ Puromycin, psiSTRIKE™ Hygromycin and psiSTRIKE™ Neomycin, also contain selectable markers for use in eukaryotic cells and can be used for both transient and stable expression of shRNA target sequences. The siSTRIKE™ U6 Hairpin Cloning System Basic and the siSTRIKE™ U6 Hairpin Cloning System hMGFP are intended for use in transient suppression assays.

The siSTRIKE™ U6 Hairpin Cloning Systems are also designed to facilitate easy determination of successful ligation. The hairpin oligonucleotides used in the siSTRIKE™ Systems are under 60bp in length. Since detection of a 60bp insert is difficult using an agarose gel, the presence of an insert can be detected by Pst I digestion. The psiSTRIKE™ Vectors contain a single Pst I site. Successful insertion of a hairpin oligonucleotide creates a second Pst I site. Therefore, digestion with Pst I will yield two DNA fragments in the presence of an insert. Pst I digestion of psiSTRIKE™ Vectors that do not contain insert will result in linearization of the vector, which will appear as a single band on an agarose gel (Figure 2.10).

siRNA Target Sequence Selection

The siRNA Designer can be used to assist in the selection of target sequences and in the design of hairpin oligonucleotides for use in the siSTRIKE™ Systems. The psiSTRIKE™ Vectors are provided in a linearized form with specific, single-stranded overhangs. The siRNA Designer will design potential target sequences that have the appropriate, complementary overhang sequences at the ends of the hairpin for efficient ligation to the psiSTRIKE™ Vectors. Two hairpin oligonucleotides must be synthesized for each target sequence tested. Standard desalting of the oligonucleotides is required prior to use; gel purification and 5′ phosphorylation are not required.

Cloning shRNAs into the psiSTRIKE™ Vectors

There are five basic steps involved in cloning a hairpin sequence using the siSTRIKE™ U6 Hairpin Cloning Systems: 1) annealing of the hairpin oligonucleotides, 2) ligation of the annealed oligonucleotides into the vector, 3) transformation into E. coli, 4) purification of DNA and 5) visualization of successful ligation products. These steps are optimized for efficient ligation and easy detection of successfully ligated hairpin sequences. The linearized psiSTRIKE™ Vectors supplied with the system eliminate the need for vector preparation. When ligating annealed hairpin oligonucleotides, we routinely observe 100-fold more colonies from ligation reactions with insert than with vector-alone control ligations (Bailey et al. 2004).

An overview of the siSTRIKE™ U6 Hairpin Cloning System protocol is given in Figure 2.11. An animated presentation illustrating the siSTRIKE™ Systems protocol is also available.

Transient and Stable In Vivo Suppression

The psiSTRIKE™ Basic and psiSTRIKE™ hMGFP Vectors are recommended for use in transient transfection assays. For many target sequences, transient transfection can quickly yield cells that can be assessed for the effects of gene suppression, including changes in phenotype, protein expression levels or other effects.

One consideration in evaluating transient transfection experiments is transfection efficiency. For example, if only 30% of the cells are transfected, it may be difficult to detect inhibition of expression, since 70% of the cells were not successfully transfected and still express the target mRNA. The siSTRIKE™ U6 Hairpin Cloning System hMGFP (Cat.# C3550) provides an shRNA expression vector that contains an internal fluorescent marker for monitoring delivery efficiency of shRNA-expressing constructs. In addition to allowing transcription of hairpin target sequences and generation of siRNAs in vivo, the vector contains an improved, synthetic version of the green fluorescent protein (hMGFP) gene. The hMGFP gene encodes a 26kDa protein that gives improved fluorescence and reduced cytotoxicity compared with other GFP proteins. The presence of GFP allows easy determination of transfection efficiency and allows selection of transfected cells by fluorescence-activated cell sorting (FACS^®, Cormack et al. 1996; Sorensen et al. 1999; Galbraith et al. 1999). Importantly, the expression of hMGFP does not affect gene silencing by shRNA molecules expressed from the same vector.

The psiSTRIKE™ Puromycin, psiSTRIKE™ Hygromycin and psiSTRIKE™ Neomycin Vectors allow selection of stably transfected cells. Thus, the results are no longer dependent on transfection efficiency. If necessary, clonal lines of transfected cells can be generated if the population of selected cells does not show the expected inhibition levels. Because integration of the vector into different positions in the genome can affect expression of the RNAi hairpin, a population of cells may not show suppression. A clonal cell line that sufficiently expresses the RNAi hairpin may be required to demonstrate suppression with the psiSTRIKE™ Vectors. Example protocols for transient and stable transfection using the psiSTRIKE™ Vectors are given in Section V.B.

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RNA Delivery Strategies

Successful RNAi experiments are dependent on both siRNA design and effective delivery of siRNA duplexes into cells. RNAi delivery strategies vary depending on the target cells or organism. For example C. elegans may be injected (Fire et al. 1998; Grishok et al. 2000), soaked in (Tabara et al. 1998), or fed (Timmons and Fire, 1998; Kamath et al. 2001; Fraser et al. 2000) dsRNA. Successful delivery of interfering RNA has also been achieved by microinjection of RNA into Drosophila embryos (Kennerdell and Carthew, 1998) and mouse oocytes (Wianny and Zernicka-Goetz, 2000). Delivery to Drosophila S2 cells in culture can be achieved by incubating the cells with the chosen RNA (Clemens et al. 2000; Betz and Worzella, 2003). Use of DNA-based approaches like ddRNAi vectors allows use of standard transfection reagents/methods, for example, cationic lipids, calcium phosphate, DEAE-Dextran, polybrene-DMSO or electroporation (Caplen et al. 2001; Elbashir et al. 2001a).

Transfection Reagents for Delivery of siRNA Duplexes

The majority of transfection reagents are optimized for delivery of plasmid DNA and not for delivery of siRNA duplexes. Therefore, new formulations have been developed to facilitate this new technology. The CodeBreaker™ siRNA Transfection Reagent (Cat.# E5052) is optimized for the efficient transfection of siRNA. The reagent promotes efficient siRNA transfer into mammalian cells, allowing siRNA-mediated gene silencing with minimal levels of cell death compared to other siRNA transfection reagents. The CodeBreaker™ Reagent is mixed with the appropriate siRNA duplex and serum-free media. The resulting complex is added directly to cultured cells. Transfection can be performed in the presence of complete growth media, eliminating the requirement for a media change. Figure 2.12 shows the CodeBreaker™ Reagent transfection protocol.

Transfection of ddRNAi Vector Constructs

Once annealed hairpin oligonucleotides or PCR products are ligated to the appropriate psiSTRIKE™ or psiLentGene™ Vector, the resulting constructs can be used for transient or stable transfection. Both the siSTRIKE™ and siLentGene™-2 Hairpin Cloning Systems provide a choice of vectors containing various selectable markers (neomycin, hygromycin or puromycin) that can be used for stable expression of a pool of cells or individual clones. Transfection of the plasmid DNA into human cells may be mediated by cationic lipids, calcium phosphate, DEAE-Dextran, polybrene-DMSO or electroporation. Transfection conditions will need to be optimized for your particular system. Guidelines for transfection of the psiSTRIKE™ and psiLentGene™ Vectors are provided in Technical Manuals #TM246 and #TM247, respectively. General considerations for transient and stable transfection are given below.

Transient Transfection

High transfection efficiency is essential for achieving substantial suppression levels using a transient transfection approach. Prior to testing for suppression of the target protein, optimize the transfection conditions for maximum efficiency in the system to be tested. The psiSTRIKE™ Basic, psiLentGene™ Basic and psiSTRIKE™ hMGFP Vectors are recommended for use in transient transfection assays. When using the psiSTRIKE™ Basic or psiLentGene™ Basic Vectors, optimization can be performed using a GFP reporter vector such as the Monster Green^® Fluorescent Protein phMGFP Vector (Cat.# E6421). The psiSTRIKE™ hMGFP Vector already contains the GFP reporter. The GFP reporter can also be used to determine transfection efficiency for each assay. This control can be performed as a separate transfection to determine the percentage of the cell population transfected or as a cotransfection where flow cytometry is used to sort GFP-positive cells. The level of target suppression in transfected cells can then be determined, taking the transfection efficiency into account.

Variations in suppression efficiency can occur depending on the cell line, cell culture conditions, target sequence and transfection conditions. Varying the amount of transfection reagent, the amount of DNA used and the cell density can influence transfection efficiency. Obtaining the highest transfection efficiency with low toxicity is essential for maximizing the siRNA interference (suppression) effect in a transient assay. Additionally, maintaining healthy cell cultures is essential for this application. The key parameters for optimization include cell density at transfection, cell proliferation and time between transfection and analysis of the RNAi effect.

Cell Density (Confluence) at Transfection: The recommended cell density for most cell types at transfection is approximately 30–50%; this level is lower than standard transfection experiments where cells are plated at 50–70% confluency. The optimal cell density should be determined for each cell type. Continued proliferation and the need to passage cells should be considered when determining the number of cells to plate.

Cell Proliferation: The successful suppression of gene expression requires actively proliferating and dividing cells, so it is essential to maintain healthy cell cultures. It is essential to minimize the decrease in cell growth associated with nonspecific transfection effects and to maintain cell culture under subconfluent conditions to assure rapid cell division. We recommend using the CellTiter-Glo^® Luminescent Cell Viability Assay (Cat.# G7570) to monitor cell viability and growth.

Time: The optimal time after transfection for analyzing interference effects must be determined empirically by testing a range of incubation times. Typically little inhibition is seen after 24 hours, but the maximal suppression time can vary from 48–96 hours depending on the cells used and the experimental targets tested.

Protocol: Transient Transfection RNAi Assay Using the psiSTRIKE™ hMGFP Vector

This protocol describes suppression of caspase-3 expression in HeLa cells using various psiSTRIKE™ hMGFP Vector constructs in a transient transfection assay. Note that this is an example protocol only. Optimization of transfection conditions is required for each particular suppression assay.

Materials Required:

• psiSTRIKE™ hMGFP Vector (Cat.# C3550) containing shRNA sequence(s)
• transIT^®-LT1 Transfection Reagent (Mirus Cat.# MIR 2304)
• cultured cells, serum-free medium and complete medium
• anti-caspase 3 antibody (Imgenex, Cat.# IMG-144)
• anti-β-actin antibody (Abcam Cat.# AC-15)
• standard gel electrophoresis and Western blotting equipment and reagents
• fluorescence microscope

1. One day before transfection, plate 3 x 10⁴ cells/ml (1ml/well ) in complete growth medium without antibiotics in a 12-well plate.

2. Add serum-free medium (250µl of medium for each well in the assay plate) to a sterile tube. Add 3µl/well Mirus transIT^®-LT1 transfection reagent (Mirus Cat.# MIR2300) dropwise to the tube. Vortex thoroughly. Incubate at room temperature for 5–15 minutes.

3. Add 0.5–0.8µg of the psiSTRIKE™ hMGFP Vector expressing caspase-3 shRNA per assay well to the tube containing transfection reagent. Mix by gentle pipetting. In addition to testing psiSTRIKE™ constructs containing shRNAs directed against the target gene, it is important to include control constructs in the experimental design. In this example, four psiSTRIKE™ constructs containing the same siRNA sequence directed against caspase-3 (previously screened for effectiveness in a psiCHECK™ Vector assay [see Section II.B]) and negative control psiSTRIKE™ hMGFP constructs containing a nonspecific shRNA sequence or shRNA directed against Renilla luciferase were also tested.

4. Incubate at room temperature for 5–15 minutes.

5. Add 250µl of the transfection reagent/DNA complex dropwise to each well of the 12-well plate containing the cells. Gently rock the plate.

6. After 24 hours, replace the original medium with fresh complete growth medium and incubate for a further 24–48 hours before assaying for suppression.

7. After 48 hours, hMGFP product was detected by fluorescence microscopy, providing a measure of transfection efficiency (Figure 2.13). Cells were collected and lysed, and caspase-3 supression was measured by Western blotting with specific antibody (Figure 2.14).

Protocol: Stable Transfection of psiSTRIKE™ and psiLentGene™ Vector Constructs

For stable expression, antibiotic selection must be applied following transfection. Cell lines vary in the level of resistance to antibiotics, so the resistance of a particular cell line must be tested before attempting stable selection. A “kill curve” will determine the minimum concentration of the antibiotic needed to kill nontransfected cells. The antibiotic concentration used for selection will vary depending on cell type and growth rate. In addition, cells that are confluent are more resistant to antibiotic selection, so it is important to keep the cells at a subconfluent level. The typical effective ranges and lengths of time needed for selection of both psiSTRIKE™ and psiLentGene™ constructs are given in Table 2.1.

For example, to generate a kill curve for G-418 selection, test G-418 concentrations of 0, 100, 200, 400, 600, 800 and 1,000µg/ml to determine the concentration that is toxic to nontransfected cells. Once the effective concentration of antibiotic has been determined, transfected cells can be selected for resistance.

1. Following transfection, seed cells at a low cell density.

2. Apply antibiotic to the medium at the effective concentration determined from the kill curve.

3. Prepare a control plate for all selection experiments by treating nontransfected cells with antibiotic in medium under the experimental conditions. This control plate will confirm whether the conditions of antibiotic selection were sufficiently stringent to eliminate cells not expressing the resistance gene.

4. Change the medium every 2–3 days until drug-resistant clones appear.

5. Once clones (or pools of cells) are selected, grow the cells in media containing the antibiotic at a reduced antibiotic concentration, typically 25–50% of the level used during selection.

Delivery of dsRNA to Drosophila S2 Cells in Culture

The protocol outlined below was used to successfully deliver PCR products of various sizes (180bp or 505bp) generated either from the 778bp ERK-A target or from a control plasmid containing the Renilla luciferase gene [phRL-null Vector (Cat.# E6231); 500bp or 1,000bp] to Drosophila S2 cells in culture (Figure 2.16; Betz and Worzella, 2003). Purified, in vitro-synthesized ERK-A dsRNA was introduced into Drosophila S2 cells using the method described by Clemens et al. (2000) following the protocol described below.

1. Incubate 1 x 10⁶ S2 cells in 1ml of Drosophila expression system (DES) serum-free medium (Invitrogen) in triplicate wells of a six-well culture dish in the presence or absence of various amounts (0, 9.5, 38, or 190nM) of the test (ERK-A) dsRNA or a nonspecific (Renilla luciferase) dsRNA.

2. Incubate the S2 cells at room temperature with the dsRNA for 1 hour, then add 2ml of complete growth medium.

3. Incubate the cells at room temperature for an additional 3 days to allow for turnover of the target protein.

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Quantitating siRNA Target Gene Expression

Reduction of the targeted gene expression can be measured by 1) monitoring phenotypic changes of the cell, 2) measuring changes in mRNA levels (e.g., using RT-PCR), or 3) detecting changes in protein levels by immunocytochemistry or Western blot analysis (Figure 2.17) (Huang et al. 2003; Kullmann et al. 2002; Lang et al. 2003). The suppression effect will vary depending on the target, cell line and experimental conditions.

Controlling for nonspecific effects on other targets is very important. As a negative control, cells can be transfected with either a nonspecific or scrambled target sequence. This will show that suppression of gene expression is specific to the expression of the hairpin siRNA target sequences. When suppression is determined by Western analysis, positive controls for other genes (e.g., tubulin or actin) should be included (Huang et al. 2003). Additional controls may also be desirable (Editorial (2003) Nat. Cell Biol. 5, 489–90).

Confirming the RNAi Effect

Several summary articles are available that suggest various options for controls that should be incorporated into RNAi experimental design to ensure accuracy and correct identification of an RNAi effect (Editorial (2003) Nat. Cell Biol. 5, 489–90; Duxbury and Whang, 2004). The preferred control is to show restoration of functionality of a gene through artificial overexpression of the target gene in a form that is not affected by RNAi. For example, the target gene can be engineered to contain silent mutations that render the mRNA invulnerable to the RNAi effect and introduced into the cell on a plasmid vector. If such constructs “rescue” the original function of the gene, this is a good indication that the observed suppression is mediated by RNAi. Use of siRNAs targeting several different areas of the same gene to suppress expression may also be used to provide evidence that an effect is mediated by RNAi. The observation of the same suppression effect using more that one target RNA can confirm that the observed effect is indeed RNAi.

For experiments using in vitro-synthesized siRNAs, the minimum concentration of RNAi showing an effect should be used to avoid nonspecific effects due to the introduction of large quantities of RNA into the cell. Ideally, any observed suppression should be confirmed at both the mRNA and protein levels. Northern blotting and quantitative, real-time RT-PCR can be used to demonstrate reduction of expression at the RNA level. Quantitative Western blotting, phenotypic and functional assays are some of the options available to show protein knockdown.

Negative and Positive Controls

Scrambled siRNAs and siRNAs containing a single mismatch can be used as negative controls. However, the latter are regarded as more informative. Positive controls with RNAs known to exhibit an RNAi effect may also be useful.

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Products may be covered by pending or issued patents. Please visit our Web site for more information. For additional patent and licensing information on the siLentGene™-2 and siSTRIKE™ Systems, see (a) through (d) below. For additional patent and licensing information on the psiCHECK™ Vectors, see statements (e) through (g), below.

^(a)Licensed under U.S. Pat. No. 6,573,099, Australia Pat. No. 743316 and related patents from Benitec Australia, Ltd., and/or Commonwealth Scientific and Industrial Research Organisation.

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^(d)This product is the subject of the following patents: U.S. Pat. No. 6,573,099; GB Pat. Nos. 2353282 and co-owned by Benitec Australia, Ltd., and exclusively licensed to Promega. Not-for-profit and academic entities who purchase this product receive the non-transferable right to use the product and its components in non-Commercial Purpose internal research. Only researchers at not-for-profit or academic entities may transfer materials/organisms made through the use of the product to scientific collaborators at other not-for-profit or academic entities, provided the transfer is not for any Commercial Purpose and said collaborator agrees not to transfer the materials/organisms to any third party and to use such materials/organisms solely for non-Commercial Purpose internal research. For all other purchasers, the purchase of this product conveys to the buyer the nontransferable right to use the product and its components in non-Commercial Purpose internal research for an eligible six (6) month period. To continue using the product, such purchasers are required to execute a commercial research license with Promega and Benitec by contacting Promega. Entities wishing to use ddRNAi as a therapeutic agent or as a method to treat/prevent human disease should contact Benitec directly for a human therapeutics license. Such purchasers cannot sell or otherwise transfer this product or components or any materials or organisms made using this product or components for any Commercial Purpose. Commercial Purpose means any activity by a party for tangible consideration and may include, but is not limited to: (i) use of the product in manufacturing; (ii) use of the product to provide services, information or data for a compensatory fee; (iii) use of the product for therapeutic, diagnostic or prophylactic purposes; or (iv) resale of the product or its components whether or not the product is resold for use in research. If buyer is not willing to accept the limitations of this label license, Promega is willing to accept the return of the product with a full refund. For terms and additional information relating to a commercial research license, go to: www.promega.com/licensing/. For licensing for any human therapeutic purpose, contact Benitec Australia, Ltd., at +61 7 3217 8540 or email: info@benitec.com.

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