Technical Considerations in the Use of RNA Interference

Read this article to learn about the technical considerations required in the use of RNA interference.

In several respects the approaches for silencing gene expression using RNAi methods are similar to those used for antisense DNA-mediated suppression of gene expression.

In principle, any cloned gene can be targeted by designing RNA oligonucleotides or RNA-expressing viral vectors with sequences complementary to the mRNA transcribed from the target gene.

Design and Synthesis of Small Interfering RNAs:

Double-stranded RNAs of 20-23 nucleotides (siRNAs), which can be synthesized in large quantities and transfected into cells, are the most commonly used reagents for RNAi in cultured cells. All that is needed to implement siRNA-mediated silencing of expression of a gene of interest is the cDNA sequence of that gene, and commercially available reagents with which to perform the synthesis.

Although targeting of siRNAs to any region of an mRNA would be expected to induce degradation of the mRNA and therefore, abolish production of the encoded protein, empirical data suggest that the probability of achieving selective silencing can be increased by targeting the siRNAs to specific regions of the mRNA. Generally following approach is recommended for designing a siRNA.

(1) Beginning with the AUG start codon of the target gene transcript, scan downstream for AA dinucleotide sequences; each AA and the 3′ adjacent 19 nucleotides are potential siRNA targets.

(2) Compare the sequences of the potential target sequences to sequences in the species-appropriate genome database  and eliminate from consideration of any tar­get sequences that are homologous to other coding sequences.

(3) Select 3-4 target sequences along the length of the gene for production of siRNAs. Of course it is important for all siRNA experi­ments to include negative control siRNAs with the same nucleotide composition but a scrambled sequence.

Chemical synthesis was the first method used to produce siRNAs, but now they can be produced in any laboratory using in vitro transcription methods. One protocol involves the synthe­sis of DNA oligonucleotides that include an 8-base sequence complementary to the 5′-end of a T7 promoter primer.

Each gene specific oligonucleotide is annealed to the T7 promoter primer, and a fill-in reaction using Klenow fragment produces a double-stranded template for use in an in vitro transcription reaction. The two RNA products of the in vitro transcription reactions are hybridized to each other, treated with DNase (to remove the DNA template) and RNase (to even the ends of the dsRNA), and the RNA is column purified.

Another protocol for the production of siRNAs takes advantage of the availability of recombinant human Dicer. Large in vitro transcribed RNA templates are cleaved by Dicer to produce multiple species of 22 base pair siRNAs. An advantage of the latter method is that, because it produces a mixture of different siRNAs directed against the same mRNA target, the probability of obtaining gene silencing is increased.

Construction of Plasmids and Viral Vectors for RNA Interference:

There are several reasons why expression plasmids and viral vectors are being used in basic and applied RNAi research. One major reason is that expression vectors allow continuous production of siRNAs in cells and, therefore, sustains depletion of the protein encoded by the targeted mRNA.

A second reason is that, particularly with viral vectors, the transfection efficiency of certain types of cells, particularly post mitotic cells, can be greatly increased. A third advantage of viral vectors is that they are typically more effective in obtaining sustained expression (and gene silencing, in the case of RNAi) in vivo.

For example, adenoviral vectors have been extensively used to express genes in post mitotic neurons in vivo (Smith and Romero, 1999). Short hairpin RNAs (shRNAs) can be transcribed from RNA polymerase III promoters in cells in culture or in vivo allowing continuous suppression of expression of the targeted mRNA (Paddison et al., 2002).

The latter authors proposed the use of this technology in the generation of transgenic mice as an alternative approach to gene knockout mice. Brummel Kamp and colleagues (2002) developed a novel vector system for the stable expression of siRNAs in mammalian cells. They used the polymerase-III H1-RNA gene promoter, which produces a small RNA transcript lacking a poly-adenosine tail and has a well-defined transcription start and termination signals.

The construct also allows cleavage of the tran­script at the second uridine after the termination resulting in a transcript that resembles the ends of synthetic siRNAs. They designed a gene-specific insert that specified a 19-nucleotide sequence de­rived from the target transcript, separated by a short spacer from the reverse complement of the same 19-nucleotide sequence resulting in the production of a 19-base pair stem-loop structure.

This vector system was shown to be effective in sustained suppression of target gene expression in several different types of cultured cells. Several laboratories have constructed plasmids that contain DNA templates for the synthesis of siRNAs under the control of the U6 promoter.

For example, Sui et al. (2002) inserted DNA fragments that acted as templates for the synthesis of small RNAs under the control of the mouse U6 promoter that directs the synthesis of a Pol Ill-specific RNA transcript to generate an RNA composed of two identical 21-nucleotide sequence motifs in an inverted orientation, separated by a 6-base pair spacer of non-homologous sequences.

Five thymidine’s that function as a termination signal for Pol III were added at the 3′-end of the repeat; the resulting RNA is predicted to fold back to form a hairpin dsRNA with a 3′ overhang of several thymidine’s. Using this plasmid, they were able to demonstrate the efficient inhibition of expression of three different endogenous genes (lamin A/C, CDK-2, and DNA methyltransferase) in cultured human cells.

Retroviral delivery systems have been developed based upon several commercially available vectors. For example, a retroviral siRNA vector was developed in which the U6 promoter and anti- target gene hairpin was sub-cloned into pMSCV puro at the unique Nsil site just upstream from the 3′ long terminal repeat (Devroe and Silver, 2002).

Using this retroviral siRNA delivery system, they demonstrated the efficient and sustained depletion of the NDR kinase and the transcriptional coactivator p75 in cultured cells. Lentiviral systems for shRNA delivery have also been developed. Lentiviruses can infect noncycling and post mitotic cells, and also provide the advantage of not being silenced during development allowing generation of transgenic animals through infection of embryonic stem cells or embryos (Naldini, 1998; Lois et al., 2002; Pfeifer et al., 2002).

Using this approach, silencing of green fluorescent protein (GFP) in GFP-positive transgenic mice has been shown after transduction with lentiviruses expressing shRNA directed against the GFP protein (Tiscornia et al., 2003). More recently, Rubinson et al. (2003) used lentivirus-delivered shRNA to induce silencing of CD8 and CD25 in cycling primary T cells and the pro-apoptotic molecule Bim in primary bone marrow-derived dentritic cells.

Lentiviral-mediated silencing of CD8 in hematopoietic stem cells was still present after injection of the cells in lethally irradiated congenic mice. Moreover, in vivo silencing for CD8 or p53 was also observed after infection of ES cells or zygotes leading to stable and functional silencing in adult RNAi transgenic mice.

Transfection Methods:

Several different transfection methods previously used to introduce oligodeoxynucleotides and DNA plasmids into cells have been used to successfully introduce siRNAs into cells. However, it has become clear there is no single transfection method that can be successfully applied to all cell types under all experimental conditions.

It is, therefore, important to optimize transfection conditions so that maximum gene silencing is achieved. The following transfection parameters have been shown to affect transfection and gene silencing efficacy; cell culture conditions, including cell density and medium composition; the type and amount of transfection agent; the quality and amount of siRNA; and the length of time that the cells are exposed to the siRNA.

For proliferating cells, a sub-confluent cell density is preferable. For post mitotic cells such as neurons, cell densities in the range of 200 to 500 cells per mm² of culture surface work well. Because proteins in serum can bind to and/or degrade siRNAs, the transfection should be performed in serum free medium.

Differences have been reported in the ability to transfect and silence gene expression between adherent and non-adherent cells. Post mitotic cells such as neurons and muscle cells tend to be more difficult to transfect using liposomes compared to mitotic cells such as stem cells, fibroblasts, and tumour cells.

Calcium phosphate-mediated transfection has been used successfully by several laboratories. The most com­monly used and effective transfection method for short-term suppression of gene expression using RNAi is to incorporate siRNAs into liposomes. There are an increasing variety of such transfection reagents including Oligofectamine, LipofectAMINE-2000, CellFectin, Effectene, si- PORT-Amine and siPORT-Lipid.

Other methods that have proven effective for transfecting siRNAs into cul­tured cells include electroporation (Calegari et al., 2002; McManus et al., 2002; Randall et al., 2003), microinjection (Calegari et al., 2002; Kim et al., 2002), and hydrodynamic shock (McCaffrey et al., 2002). Similar transfection methods have been used to introduce RNA-expressing plasmids into cultured cells (Iratni et al., 2002; Czauderna et al., 2003).

These studies also demonstrated the therapeutic potential of RNAi. The methods used to transfect cells with viral vectors that produce shRNAs are essentially identical to those used to transfect cells with similar vectors designed to express cDNAs (Abbas-Terki et al., 2002; Barton and Medzhhitov, 2002; Devroe and Silver, 2002; Xia et al., 2002).

It should be recognized that as RNAi technology advances it will likely be possible to produce RNAi “knockout” mice (or other mammals) in which the expression of a protein of interest is repressed by the expression of its corresponding RNAi related molecule (shRNA or miRNA, for example). The resulting animal could be seen as an equivalent of its knockout generated by targeted gene disruption but with much more flexibility and efficiency.

For example, cell type-specific promoters could be used to effect PTGS only in cells of interest; in many cases this may circumvent embryonic lethality resulting from gene deletion from all cells. It would also be quicker and less costly to produce RNAi transgenic animals compared with conventional knockouts.