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Expression Arrest Lentiviral shRNAmir: Mammalian RNAi Comes of Age

Gwen D. Fewell, Ph.D.*, and Rusla M. DuBreuil, Ph.D., Open Biosystems

The efficacy of RNA interference (RNAi) for mammalian gene silencing is dependent on development of specific and versatile RNAi triggers that enable transient, stable, and in vivo inducible applications. Lentiviral shRNAmir triggers from Open Biosystems overcome several limitations inherent in siRNA and first-generation shRNA triggers. shRNAmir triggers produce more effective, specific knockdown, and shRNAmir constructs expressed from lentiviral vectors offer advanced delivery options in vitro and in vivo. Lentiviral shRNAmir triggers expressed from Polymerase (Pol) II promoters significantly advance RNAi screening applications as well as the potential for the creation of in vivo animal models. Multiplexed (pooled) RNAi positive and negative selection screens are made possible by unique molecular barcodes incorporated into the lentiviral shRNAmir vector.

Introduction

Gene silencing using RNAi has revolutionized biology and offers numerous applications for basic research and drug discovery. Discovered as a biological response to double-stranded RNA (dsRNA) in the nematode Caenorhabditis elegans (1), this evolutionarily conserved, genetic surveillance mechanism results in the sequence-specific posttranscriptional down-regulation of target genes (2,3). Since its discovery, RNAi has rapidly become a powerful tool for perturbing gene function and has accelerated both small-scale gene characterization studies as well as genome-scale screening in vitro (4,5). The ability to modulate gene expression using RNAi enables the evaluation of gene function at different levels. Rapid advances in the understanding of endogenous RNAi pathways have fueled the development of synthetic RNAi triggers and expanded delivery options in vitro and in vivo. These new-generation RNAi triggers contribute to applications for whole-genome loss of function screens and more complex genetics in animal models. RNAi continues to show great promise as a tool in biological research and as an approach for silencing disease-causing genes and human therapy.


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Evolution of Synthetic Silencing Triggers for Mammalian RNAi

siRNA and First-Generation shRNA

Three types of synthetic small RNA have been developed to perform RNAi in mammalian cells. Short interfering RNA (siRNA) was the first silencing trigger used successfully in mammalian cells, to transiently knockdown the expression of target genes (6) (Figure 1). This transient-only knockdown lasting from three to five days in culture makes the siRNA approach unsuitable for analysis of the long-term and downstream effects of gene silencing. Other limitations associated with siRNAs are the variability of transfection efficiencies in different cell lines. Many cell lines including primary and nondividing cells are difficult to transfect at the high efficiencies required to elicit knockdown phenotypes. siRNAs also have very limited functionality in vivo.

The discovery of microRNA (miRNA), endogenous triggers of the RNAi pathway, resulted in the development of another generation of silencing triggers called short hairpin RNA (shRNA), modeled after miRNA hairpin precursors and expressed from DNA vectors (7,8). First-generation shRNA triggers are transcribed under the control of RNA Polymerase III (Pol III) promoters (9,10). shRNAs are produced as single-stranded molecules of 50-70 nucleotides in length, form stem loop structures, exit the nucleus, are cleaved at the loop by the nuclease Dicer, and enter the RISC complex as si-RNAs (Figure 2). These first-generation shRNA triggers expressed from vectors containing selectable markers ensure the stable expression of shRNA and prolonged silencing of the target gene (Figure 1). The problems with these shRNA are essentially twofold, first Pol III promoters, unlike Pol II, do not lend themselves to regulation, and second, shRNAs can be ineffective inhibitors of their target mRNA when expressed at single copy.

shRNAmir Design: Increased Processed siRNA and Knockdown Efficiency

As the understanding of microRNA biogenesis advanced (7,11-14), second-generation shRNAmir triggers were developed. shRNAmir constructs are expressed as primary-miRNA (pri-miRNA) transcripts (Figure 2). These constructs were created by redesigning the most studied microRNA, human miR30, to express an artificial siRNA/miRNA. The stem of the primary microRNA-30 transcript was substituted with gene-specific sequences against different target genes. This does not perturb miRNA-30 maturation (15,16) and allows normal microRNA processing to produce mature siRNAs (Figure 2). In this way, shRNAmir derivatives of primary miR30 can target any mRNA for RNAi.

The shRNAmir design allows the addition of a Drosha cleavage site, harnessing endogenous processing by Drosha, which has been shown to increase subsequent Dicer recognition and specificity (17). shRNAmir triggers enter the RNAi pathway ahead of either siRNA or shRNA and are processed by both Drosha and Dicer, leading to more siRNAs being produced in the cell available for incorporation into the RISC complex for target mRNA degradation (15-18). This design has recently been shown to produce 12-fold greater processed siRNA as well as consistent and greater knockdown efficiencies when compared with first-generation shRNA (15). There is also some evidence that Dicer processing via the endogenous pathway results in active loading of the RISC complex (19).

Lentiviral Vectors and RNAi

shRNA and shRNAmir expressed from viral vectors for RNAi delivery take the existing technology to an advanced level, as viral vectors can be used in both transfection or infection formats for delivery. Viral infection or transduction (if a self-inactivating vector is used) is advantageous over transfection, as the integration efficiency is much greater.Viral vectors used for RNAi delivery include a packaging signal (ψ) and regulatory elements that enable packaging of the genetic elements between the long terminal repeats (LTRs). Self-inactivating retroviral vectors expressing shRNAmir constructs are already available from Open Biosystems (11,20). Retroviruses, however, only integrate into dividing cells and so are limited in their delivery into primary and nondividing cells. Lentiviruses (pseudotype VSV-G) infect a wide variety of mammalian cells with high efficiency (21) and therefore overcome the delivery limitations faced by siRNA triggers. They offer the option for infection-based delivery into most cell lines including hard-to-transfect cells such as primary and nondividing cells (22,23). Lentiviral vectors in combination with second-generation shRNAmir design thus offer a superior tool for RNAi studies. The lentiviral shRNAmir vector (Figure 2) developed in collaboration with Hannon and Elledge (22) contains several elements that make it the vector of choice for RNAi studies. These include:

Expression of the shRNAmir by a RNA Pol II Promoter

Recent studies (22,24) showed that the increased transcription of shRNAmir from Pol II over Pol III promoters is more than sufficient for highly effective knockdown, even when present at single copy in the cell. This feature of achieving knockdown at single copy is essential for screens using complex (pooled) libraries where one wants to ensure that a given cell harbors only a single shRNA construct. The demonstration of a functional Pol II promoter driving shRNAmir expression also makes it possible to consider a regulatable system, since Pol III promoters do not lend themselves to regulation.

Molecular barcodes, 60-nucleotide sequences unique to each vector, allow the abundance of each shRNAmir vector to be monitored within a complex mixture by microarray analysis. This strategy has been used successfully in lower eukaryotes and recently also in a mammalian positive selection screen (25). It is also amenable to growth- assessment assays or screening for synthetic lethal relationships (26,27). The latter types of screens are only feasible if each shRNAmir integrant demonstrates a high penetrance of the phenotype; otherwise, the dynamic range of the signal change will be too low for statistical significance and possibly detection.

GFP and the shRNAmir are incorporated into a bicistronic transcript, thereby allowing the tagging of shRNAmir-expressing cells. See Figure 3 for an example of transduction with the lentiviral shRNAmir vector showing eGFP expression as a measure of shRNAmir expression. This feature allows one to identify cells that received and express the shRNAmir construct within a complex population of cells, a feature particularly valuable for in vivo animal studies. Another important feature is the ability to flow-sort for GFP-positive cells, which obviates the need for the time-consuming generation of individual clonal isolates in many experimental settings. Figure 4 shows an example of knockdown using the lentiviral shRNAmir construct to EG5.

Inducible RNAi Systems

Given the tremendous need for tools allowing fast and efficient evaluation of gene function, drug-inducible control of gene expression in mammalian systems, especially in vivo-based RNAi, will rapidly become invaluable to the research community. Currently available systems for conditional gene inactivation often have limited in vivo functionality because of “leakiness” and insufficient level of knockdown and induction. shRNAmir constructs under the control of a Pol II promoter can produce stable and regulatable gene knockdown in cultured cells and in animals (24). A tightly regulated
shRNAmir construct based on a tetracycline-responsive promoter system (regulated by changing doxycycline levels) was directed against Trp53 shown to switch cultured mouse fibroblasts between a proliferative and senescent state. Tumors induced by Trp53 and suppression (and other cooperating oncogenes) regressed upon re-expression of Trp53. These experiments indicate that shRNAmir constructs under the control of Pol II promoters will be suitable for a variety of in vivo applications, including tissue-specific knockdowns and in vivo forward genetic screens.

Conclusion

Genomewide lentiviral shRNAmir libraries incorporate advances in both shRNA design and molecular barcode technology and enable large-scale RNAi screens that rely on subtle changes in fitness levels of cells under various environmental conditions. Such screens can even be carried out using lentiviral shRNAmir libraries from Open Biosystems that perform effectively at a single copy level. Further advances will include inducible RNAi libraries allowing the progression to more complex genetics in animal models. These advancements will likely lead to the identification of novel targets and therapeutic strategies based on new insights into complex genetic pathways.

*Address correspondence to Gwen D. Fewell, Ph.D., Open Biosystems. E-mail: gwen.fewell@openbiosystems.com.

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Figure 3. Transduction of HEK293 cells with the lentiviral shRNAmir vector. eGFP expression visualized on the right indicates cells that are expressing the shRNAmir.


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Figure 4. Silencing of the human EG5 (KIF11) gene using a shRNAmir against EG5 in HEK293T cells. The cells were transduced using an EG5 lentiviral shRNAmir and stained for DNA (DAPI, blue), tubulin (anti-tubulin, green), and EG5 (anti-EG5, red) 48 hours later. EG5 knockdown results in disruption of normal cell division and causes the formation of half spindles. Cells transduced with EG5 shRNA are arrested in mitosis and show monoastral microtubular arrays. By contrast, control cells show normal bipolar spindles and microtubule networks in mitosis and in interphase (cell on the bottom left).

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