Ser persona a lo largo de la historia de la filosofía.

1.4.1 Introduction to shRNA screens

High throughput RNAi screens have proved a powerful tool in identifying genes involved in diverse biological processes. The original screen structure tended to involve individual construct transfection of siRNAs in a 96-well format. Such screens have been enormously informative in Drosophila and other lower organisms, as well as mammalian systems. More recently, virally delivered, shRNA based libraries have allowed the development of massively parallel functional RNAi screens. This approach has identified existing and novel genes important to embryonic (Hu, Kim et al. 2009; Chia, Chan et al. 2010;

Westerman, Braat et al. 2011) and haematopoietic (Ali, Karlsson et al. 2009; Hope, Cellot et al. 2010; Baudet, Karlsson et al. 2012) stem cell maintenance, neuronal synaptic development (Valakh, Naylor et al. 2012) and circadian clock modification (Zhang, Liu et al. 2009).

shRNA based functional screens are most obviously suited to identification of genes involved in cell proliferation and survival, and have therefore become an important tool in cancer research.

The general procedure for lentiviral based screening involves:

1. Transduction of target cells with lentiviral pools to give one construct per cell;

2. Harvesting of sample to provide a baseline for construct prevalence; 3. Period of culture to determine effect of gene knockdown, combined

with antibiotic selection to remove untransduced cells; 4. Harvesting second sample for comparison with baseline.

The effect of knockdown of individual genes can be assessed by analysing the variation in construct prevalence between the baseline sample taken prior to knockdown and the second sample taken after a period of culture with

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1.4.2 Screening approaches – Positive and Negative screening

In broad terms, screens can be conducted to produce either positive selection or negative selection outputs. The former involves an assay in which the output is colonies which have been positively selected following exposure to an

environmental pressure (e.g. irradiation, drug treatment, immunophenotypic selection etc) (Westbrook, Martin et al. 2005; Brummelkamp, Fabius et al. 2006; Gazin, Wajapeyee et al. 2007). This approach requires sequencing of

individual clones and provides information only on genes deleterious to survival under selective pressure (constructs targeting these genes will be identified from proliferating clones). Negative selection involves analysis of the whole population at the end of the assay and therefore provides information on the prevalence of constructs relative to each other, including those which have become more, and those which have become less, prevalent (Schlabach, Luo et al. 2008; Silva, Marran et al. 2008). Information is therefore gathered on genes which are beneficial to cell growth and survival (in this instance, hairpin construct prevalence will decrease) as well as those which are deleterious.

1.4.3 The development of shRNA libraries

In order to identify the causative construct of a phenotype generated by a pooled/parallel RNAi screen, it is essential that: 1) each cell should receive only a single construct; 2) individual constructs can be identified at the end of the experiment.

The first constraint was addressed by development of microRNA adapted hairpins by the laboratories of Greg Hannon, Cold Spring Harbour Laboratories, and Stephen Elledge, Harvard University (Dickins, Hemann et al. 2005; Silva, Li et al. 2005; Stegmeier, Hu et al. 2005). By cloning the hairpin into the middle of a sequence derived from miRNA-30 (mir30), the construct is believed to follow a more natural pathway from transcription to RISC via processing by DROSHA and DICER. This results in more efficient processing, active export from the nucleus and active loading into RISC, see Figure 1-4. A substantially greater knockdown is achieved, making just a single lentiviral integration effective. The second of these requirements has been satisfied by sequencing or array based analysis of a specific construct. The two principle approaches available are the analysis of either a barcode, unique to an individual shRNA construct, or analysis of the unique construct itself. Modern iterations of the second option tend to rely on analysis of a “half-hairpin” to avoid probe self-annealing during array analysis (Schlabach, Luo et al. 2008; Silva, Marran et al. 2008).

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1.4.4 Limitations of shRNA screens

Genome-wide screens are so complex, that is to say they require accurate transduction of a very large number of constructs with a high degree of

coverage, that to perform them in primary material is unfeasible. This presents a difficulty, as hits identified by the in vitro analysis may not remain valid in an in vivo situation. In a head-to-head comparison of in vitro and in vivo screening, Meacham et al found less than 10% of differentially represented hairpin

constructs to be shared between the two situations (Meacham, Ho et al. 2009). Another difficulty with the complexity of whole-genome screens is that

constructs have not been prospectively validated. Instead, hairpin sequences are derived from algorithms which are designed to maximise knockdown whilst minimising off-target effects, especially those derived from the critical seed- region, see 1.3.2. Despite this, it is presumed that a proportion of hairpins will not achieve substantial levels of knockdown, and it has been demonstrated that at least 1% of hits in an siRNA screen is an off-target effect (Schultz,

Marenstein et al. 2011). Recently, an analysis tool for predicting off-target effects from the seed-region has been described (Sigoillot, Lyman et al. 2012). Such approaches may help to limit false positive hits generated by off-target effects, although it may still be prudent to confirm that potential therapeutic targets are indeed false hits.

Finally, a small number of studies have demonstrated an innate toxicity to virally delivered shRNAs in the setting of murine models of disease. This effect can be modulated by expressing shRNAs from a miRNA context (McBride,

Boudreau et al. 2008). Use of miRNA adapted hairpins is not without

complications however. Two studies have shown the potential for shRNAmir constructs, which produce much higher levels of shRNA precursors than do native shRNAs, to saturate the endogenous RNAi machinery, probably at the level of nuclear export by Exportin 5, resulting in disturbed endogenous miRNA function (Grimm, Streetz et al. 2006; Pan, de Ruiter et al. 2011).

1.4.5 shRNA screening in cancer

The unbiased nature of genome-wide functional screening has allowed the identification of novel tumour-suppressor genes (Westbrook, Martin et al. 2005; Zender, Xue et al. 2008; Iorns, Ward et al. 2012) as well as potential therapeutic targets (Cole, Huggins et al. 2011; Zuber, Shi et al. 2011). RNAi screens can also be adapted to more complex assays. Within cancer research this includes synthetic lethality screens used to look for factors potentiating the effect either of an existing therapy (Azorsa, Gonzales et al. 2009), or of a commonly mutated gene such as RAS (Luo, Emanuele et al. 2009) or BRCA2 (Hattori, Skoulidis et al. 2011). The standard cellular survival screen can also be adapted to look at specific cell populations and has been applied to candidate brain tumour stem cell populations to identify genes essential for cancer stem cell survival and maintenance (Wurdak, Zhu et al. 2010; Goidts, Bageritz et al. 2012).

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