Authors: Gustavo Tiscornia, Oded Singer and Inder M. Verma
This protocol was adapted from “Development of Lentiviral Vectors Expressing siRNA,” Chapter 3, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.
This protocol combines the specificity of small interfering RNA (siRNA)-mediated silencing cassettes with the versatility of lentiviral vectors to stably transduce a wide range of cell types. A short hairpin RNA (shRNA) designed against a given target is cloned into a plasmid containing the pol III promoter. The design uses a 5′ forward primer upstream of the pol III promoter and a 3′ reverse primer that includes the entire shRNA sequence (i.e., sense, loop, and antisense sequences followed by five Ts), followed by 22 bases complementary to the last 22 bp upstream of the +1 transcriptional start site of the pol III promoter. An NheI-compatible restriction site is included at the 5′ end of both forward and reverse primers. A single round of PCR is used to amplify this template. The resulting DNA fragment contains an shRNA expression cassette that can be cloned into a simple cloning vector, tested, and then transferred to the lentiviral vector, or cloned into the lentiviral vector directly. This procedure uses a unique restriction site in the 3′ long terminal repeat (LTR). During integration, the 5′ LTR of the provirus is copied from the 3′ LTR, cloning the H1-driven shRNA into the 3′ LTR, resulting in duplication of the silencing cassette. This strategy maximizes the silencing power of the lentiviral vector. The combination of the lentiviral and siRNA technologies provides a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo.
Figure 1 presents a schematic representation of the protocol. One undesirable consequence of this procedure is that the siRNA target sequence is also present in the mRNA expressing the marker gene, resulting in somewhat lower expression of the marker. In an alternative method, Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B (Tiscornia et al. 2008), the position of the silencing cassette is upstream of the marker expression cassette, thus avoiding down-regulation of the marker. Because the silencing cassette is not in the 3′ LTR, only one copy of the silencing cassette is delivered per viral particle.
Figure 1. The siRNA expression cassette consists of a nucleotide sense sequence (identical to the target sequence in the mRNA to be down-regulated), followed by a 9-bp loop, an antisense sequence, and a stretch of five Ts (T5) as a pol III transcriptional termination signal downstream from an H1 promoter. After PCR amplification, this is packaged into the NheI site of a transfer vector containing all the cis-acting elements required for replication and packaging of RNA into viral particles, including a Rev-responsive element (RRE) to enhance nuclear export of unspliced viral genomic RNA, woodchuck hepatitis virus regulatory element (WPRE) to enhance expression of the transgene, and a central polypurine tract (cPPT) purported to increase efficiency of nuclear import of the preintegration complex. Green fluorescent protein (GFP) is used as a marker.
A number of algorithms have been developed to predict effective siRNA sequences (e.g., http://www.ambion.com/ or http://sfold.wadsworth.org/). A database search is recommended to filter out candidate targets that are present in other genes to avoid silencing of these loci.
Design and Cloning of shRNAs
This results in an amplified fragment of ~400 bp that can be cloned in an A/T vector for sequencing or directly cloned in the lentivector plasmid.
4.Digest the insert with XbaI and gel-purify.
5.Digest the lentivector plasmid with NheI, gel-purify, and then dephosphorylate.
6.Validate the cloned shRNA cassettes by transfecting or transducing (as lentiviral particles) to a cell line that expresses the target gene.
7.Alternatively, coexpress a tagged cDNA of the target gene together with shRNA silencing cassettes in an easily transfected cell line (e.g., 293T).
Preparation of Lentiviral Vectors
8.Twenty-four hours before transfection, prepare plates and cells:
9.Transfect the plasmid mix into the cells using the CaPO4 precipitation method:
10.Approximately 16-20 h after transfection, remove the media. Add 15 mL of fresh DMEM with 2% FBS to each plate. Incubate in a 10% CO2 atmosphere overnight at 37°C.
11.Collect the supernatant from the plates. Filter through 0.22- or 0.45-μm filters. Add 15 mL of fresh medium to each plate and incubate overnight.
12.Collect media from the plates and filter as in Step 11.
13.Pool collected supernatants from Steps 11 and 12. Transfer to 30-mL centrifuge tubes, using 25-29 mL per tube. Concentrate the viral particles by centrifuging in an SW 28 rotor at 19,400 rpm for 2 h at 20°C.
14.Resuspend all pellets in a total of 1 mL of HBSS. Wash tubes a second time with 1 mL of HBSS.
15.Add HBSS to the resuspended pellets and tube washing fluid to a final total volume of 3 mL.
16.Prepare a 1.5-mL cushion of 20% sucrose in HBSS in 5-mL centrifuge tubes. Layer the resuspended pellets on the sucrose cushion. Centrifuge using an SW 55 rotor at 21,000 rpm for 1.5 h at 20°C.
17.Resuspend the pellet in 100 μL of HBSS. Wash the tube with an additional 100 μL of HBSS.
18.Shake the resuspended viral preparation on a low-speed vortexer for 15-30 min.
19.Centrifuge for 10 sec to remove debris. Aliquot the cleared viral solution.
20.Titrate the viral preparations by quantitating levels of the capsid protein p24 using a p24 ELISA kit (or by biological titration if an adequate marker is contained in the lentivector).