RICS complex, the effector of RNAi silencing is a multi-protein complex of which several components were identified. One of the proteins identified in almost all organisms is AGO protein that is essential for mRNA silencing activity [74]. In plants 10 AGO member proteins have been identified. For example, AGO1 mutant plants have been found to develop distinctive developmental defects. miRNAs are accumulated in these mutants but the cleavage of target mRNA not longer occur [75]. AGO4 has role in the production of long siRNAs of 24bp and it was early reported that AGO4 is involved in long siRNA mediated chromatin modifications (histone methylation and non-CpG DNA methylation) [76]. In addition to AGO family members, several other proteins associated with RISC complex have been identified in vertebrate and invertebrate models. For example, the *Drosophila*  homologue of the fragile X mental retardation protein (FMRP); R2D2, found in *Drosophila*  and thought to facilitate the passage of the Dicer substrate to the RISC; members of the mammalian Gemin family, some of which are thought to have helicase activity [44].

RNAi Towards Functional Genomics Studies 77

scientists can quickly and easily reduce the expression of a particular gene in mammalian and plant cell systems, often by 90% or greater, to analyze the effect that gene has on cellular

In plants, many efforts were concentrated on the improvement of the nutritional content using the classical breeding approaches such as selection of the natural or induced genetic variations, or by genetic engineering of transgenic plants [83]. Genetic engineering technologies have advantages over classical breeding not only because they increase the scope of genes and the types of mutation that can be manipulated, but also because they have the ability to control the spatial and temporal expression patterns of the genes of interest [84]. The delivery of siRNAs in plants has been always achieved by expressing hairpin RNAs that fold back to create a double-strand region that will be recognized by the Dicer-like enzyme. Figure 2 depicted an typically RNAi construct in plants with the promoter region, the inverted repeats of the target gene with the appropriate orientation, the spacer region which separate the two inverted repeats sequences and the terminator region.

**Figure 2.** A schematic representation of a RNAi vector cassette with the promoter/ terminator region, target inverted repeats, intron spacer region; the arrows represent the direction of transcription

**Intron spacer**

**Promoter Terminator**

 **Target gene sequence**

**Target gene sequence**

The double stranded RNA generated through aberrant gene expression from a foreign gene, viral infection, tandem repeat sequences formed due to insertion of a transposon/retrotransposon, are recognized by Dicer and digested in small interfering RNAs, which functioned as template for the targeted degradation of mRNA in RISC. This siRNA functioned also as primer for RdRP to amplify secondary dsRNA. On the other hand, in plants, RNAi is both systemic and heritable and siRNAs move between cells through channels in cell walls, thus enabling communication and transport throughout the plant. In addition, methylation of promoters targeted by RNAi confers heritability, as the new

Genetic transformation via *Agrobacterium* or by particle bombardment or by infecting plants with viruses that can express the dsRNAs, or the infiltration of *Agrobacterium* harboring the hairpin cassette for transient gene silencing are the common methods for inducing gene silencing in plant system. The transgene expression should be evaluated as soon as possible for each transgenic event, and over multiple generations to insure that each line is stablesilencing its target. Many transgenic events should be generated and analyzed and the lines with active transgenes that are effectively inducing silencing cal be selected and maintained.

methylation pattern is copied in each new generation of the cell [85].

**3.1. Development of efficient RNAi vector cassettes**

function [18, 49, 82].

#### *2.5.3. RNA-directed RNA polymerase (RdRP)*

In both plants and *C. elegans*, RNAi/PTGS requires proteins similar in sequence to a tomato RNA-directed RNA polymerase [77]. In *Arabidopsis*, RdRP homologue SDE1/SGS2 is required for transgene silencing, but not for virally induced gene silencing [78]. This may suggest that SDE1/SGS2 act as an RdRP, since viral replicases could substitute for this function in VIGS. In *Neurospora*, RdRP homologue QDE-1 is required for efficient quelling [79]. EGO-1, one of the *C. elegans* RdRP, is essential for RNAi in the germline of the worm [80], and another RdRP homologue, RRF-1/RDE-9, is required for silencing in the soma. All RdRP proteins could be involved in amplifying the RNAi signal. However, only the tomato and *Neurospora* enzymes have been demonstrated to posses RNA polymerase activity, and biochemical studies are required to establish definitively the role of these proteins in RNAi [81].

#### *2.5.4. Putative helicase*

Other proteins, helicases have been identified in plants (e.g *sde3* in *Arabidopsis*) [78]. Although possible roles in RNAi for some of these proteins were proposed, e.g. MUT6 might involved in the degradation of misprocessed aberrant RNAs [81], their functions are mostly unknown and further biochemical experiments are needed to reveal their exact roles in RNAi. The quelling-defective mutant in *Neurospora*, *qde3*, was cloned and the sequence encodes a 1,955 amino acid protein. This protein shows homology with the family of RecQ DNA helicases, which includes the human proteins for Bloom syndrome and Werner syndrome.

#### **3. Applications of RNAi in plant systems**

RNAi has been used as new tool to reduce the expression of a particular gene in mammalian and plant cell systems, to analyze the effect that gene has on cellular function, and also it has the potential to be exploited therapeutically and clinical trials. However, by using RNAi, scientists can quickly and easily reduce the expression of a particular gene in mammalian and plant cell systems, often by 90% or greater, to analyze the effect that gene has on cellular function [18, 49, 82].

#### **3.1. Development of efficient RNAi vector cassettes**

76 Functional Genomics

*2.5.2. RISC complex*

*2.5.4. Putative helicase*

RICS complex, the effector of RNAi silencing is a multi-protein complex of which several components were identified. One of the proteins identified in almost all organisms is AGO protein that is essential for mRNA silencing activity [74]. In plants 10 AGO member proteins have been identified. For example, AGO1 mutant plants have been found to develop distinctive developmental defects. miRNAs are accumulated in these mutants but the cleavage of target mRNA not longer occur [75]. AGO4 has role in the production of long siRNAs of 24bp and it was early reported that AGO4 is involved in long siRNA mediated chromatin modifications (histone methylation and non-CpG DNA methylation) [76]. In addition to AGO family members, several other proteins associated with RISC complex have been identified in vertebrate and invertebrate models. For example, the *Drosophila*  homologue of the fragile X mental retardation protein (FMRP); R2D2, found in *Drosophila*  and thought to facilitate the passage of the Dicer substrate to the RISC; members of the

mammalian Gemin family, some of which are thought to have helicase activity [44].

required to establish definitively the role of these proteins in RNAi [81].

which includes the human proteins for Bloom syndrome and Werner syndrome.

**3. Applications of RNAi in plant systems**

In both plants and *C. elegans*, RNAi/PTGS requires proteins similar in sequence to a tomato RNA-directed RNA polymerase [77]. In *Arabidopsis*, RdRP homologue SDE1/SGS2 is required for transgene silencing, but not for virally induced gene silencing [78]. This may suggest that SDE1/SGS2 act as an RdRP, since viral replicases could substitute for this function in VIGS. In *Neurospora*, RdRP homologue QDE-1 is required for efficient quelling [79]. EGO-1, one of the *C. elegans* RdRP, is essential for RNAi in the germline of the worm [80], and another RdRP homologue, RRF-1/RDE-9, is required for silencing in the soma. All RdRP proteins could be involved in amplifying the RNAi signal. However, only the tomato and *Neurospora* enzymes have been demonstrated to posses RNA polymerase activity, and biochemical studies are

Other proteins, helicases have been identified in plants (e.g *sde3* in *Arabidopsis*) [78]. Although possible roles in RNAi for some of these proteins were proposed, e.g. MUT6 might involved in the degradation of misprocessed aberrant RNAs [81], their functions are mostly unknown and further biochemical experiments are needed to reveal their exact roles in RNAi. The quelling-defective mutant in *Neurospora*, *qde3*, was cloned and the sequence encodes a 1,955 amino acid protein. This protein shows homology with the family of RecQ DNA helicases,

RNAi has been used as new tool to reduce the expression of a particular gene in mammalian and plant cell systems, to analyze the effect that gene has on cellular function, and also it has the potential to be exploited therapeutically and clinical trials. However, by using RNAi,

*2.5.3. RNA-directed RNA polymerase (RdRP)*

In plants, many efforts were concentrated on the improvement of the nutritional content using the classical breeding approaches such as selection of the natural or induced genetic variations, or by genetic engineering of transgenic plants [83]. Genetic engineering technologies have advantages over classical breeding not only because they increase the scope of genes and the types of mutation that can be manipulated, but also because they have the ability to control the spatial and temporal expression patterns of the genes of interest [84]. The delivery of siRNAs in plants has been always achieved by expressing hairpin RNAs that fold back to create a double-strand region that will be recognized by the Dicer-like enzyme. Figure 2 depicted an typically RNAi construct in plants with the promoter region, the inverted repeats of the target gene with the appropriate orientation, the spacer region which separate the two inverted repeats sequences and the terminator region.

**Figure 2.** A schematic representation of a RNAi vector cassette with the promoter/ terminator region, target inverted repeats, intron spacer region; the arrows represent the direction of transcription

The double stranded RNA generated through aberrant gene expression from a foreign gene, viral infection, tandem repeat sequences formed due to insertion of a transposon/retrotransposon, are recognized by Dicer and digested in small interfering RNAs, which functioned as template for the targeted degradation of mRNA in RISC. This siRNA functioned also as primer for RdRP to amplify secondary dsRNA. On the other hand, in plants, RNAi is both systemic and heritable and siRNAs move between cells through channels in cell walls, thus enabling communication and transport throughout the plant. In addition, methylation of promoters targeted by RNAi confers heritability, as the new methylation pattern is copied in each new generation of the cell [85].

Genetic transformation via *Agrobacterium* or by particle bombardment or by infecting plants with viruses that can express the dsRNAs, or the infiltration of *Agrobacterium* harboring the hairpin cassette for transient gene silencing are the common methods for inducing gene silencing in plant system. The transgene expression should be evaluated as soon as possible for each transgenic event, and over multiple generations to insure that each line is stablesilencing its target. Many transgenic events should be generated and analyzed and the lines with active transgenes that are effectively inducing silencing cal be selected and maintained.

Currently, several vectors used for RNAi silencing that make use of *Agrobacterium* mediated delivery or artificially introduced dsRNA and/or VIGS into plants has been reported. For example, in 2007, an *Arabidopsis* genomic RNAi knock-out line analysis consortium was lunched out (AGRIKOLA) which is using the PCR products to generate gene-specific RNAi constructs for each *Arabidopsis* gene used in large scale gene silencing studies [86-87]; other consortium called CATMA (Complete *Arabidopsis* Transcriptome MicroArray), is generating gene sequence tags (GSTs) representing each *Arabidopsis* gene, designed so that they will hybridize on *Arabidopsis* cDNA microarrays in a gene-specific manner; the *Medicago truncatula* RNAi database (https://mtrnai.msi.umn.edu/) is a NSF-funded project planning to silence 1500 genes involved in symbiosis in this model legume; amiRNAi Central (http://www.agrikola.org) a NSF project funded to provide a comprehensive resource for knockdown of *Arabidopsis* genes.

RNAi Towards Functional Genomics Studies 79

region of the target gene ensures that a specific member of a multiples gene family can be silenced. For example, RNAi can down-regulate specific target sequences when 3'UTR region is used as a trigger sequence [89-90]. It has been also shown that RNAi facilitates the generation of dominant loss-of-function mutation in polyploidy plants, even with short

Nowadays, numerous projects are lunched to produce siRNAs that will silence essential genes in insects, nematodes and pathogens using an approach called hdRNAi (hostdelivered RNAi) [92-94] based on the partial sequences similarities between plant and animal genes. There is also a limitation of this approach because, some unexpected genes

RNAi has been also used for the improvement of nutritional value of some important crops. For example, to decrease the levels of natural toxins in food plants a stable, heritable and distinct siRNA against the toxin could be used. Cottonseeds are rich in dietary proteins but unpalatable by humans as they contain a natural toxic terpenoid item, called gossypol. RNAi mechanism has been used to minimize the levels of delta-cadinene synthase, an

RNAi technology has been also applied to barley for developing varieties resistant to BYDV (barley yellow dwarf virus) [96]. In rice, RNAi has been used to reduce the level of glutein and produce rice varieties with low-glutein content [97]. Soybeans can be engineered to produce oil with low levels of polyunsaturated fatty acids through a reduction of FAD2, a fatty acyl Δ12 desaturase. This enzyme converts the monounsaturated fatty acid oleic acid (18:1Δ9) to linoleic acid (18:2Δ9, Δ12), which can be subsequently desaturated to α-linolenic acid (18:3Δ9, Δ12, Δ15) by FAD3 [98]. The reduced polyunsaturated fatty acid levels from >65% of the total oil content in normal soybean oil to less than 5% was observed [99]. In an attempt to specifically target FAD2-1, and not related family members, the soybean FAD2-1A intron was tested as an RNAi trigger, resulting in a reduction in polyunsaturated fatty acids in the seeds to about 20% [100]. This result was surprising, given that intron sequences are removed from precursor mRNAs (pre-mRNAs) by splicing in the nucleus and spatially separated from the cytoplasm

can be silenced with consequences on the organisms itself but also environment.

where mature mRNAs are presumed to be targeted by the PTGS machinery [101].

There are also some limitations of using RNAi in functional genomics studies. Unlike the insertional mutagenesis, for the use of RNAi the exact sequence of the target gene is required. Secondly, the methods to delivery RNAi are very important, some species are easily transformable and some not. Nonetheless, further improvement of the delivery methods and vectors that can be used safely and reliably are needed. There have also been some reports that revealed the difficulty to detect mutants with subtle changes in gene expression. However, in plants, numerous marker genes are being developed that will

RNA silencing in plants prevents viral accumulation and accordingly, viruses have evolved several strategies to counteract the defense mechanism. A viral protein, HC-Pro (helper

dsRNAs of a 37 nucleotide long [91].

enzyme crucial for the production of gossypol [95].

indicate if a change in gene expression occurs [102].

**3.3. RNAi and viral infections**

Moreover, a set of binary vectors, called ChromDB's RNAi vectors, were designed for producing dominant negative RNAi mutants using a target sequence cloning strategy that is based on the inclusion of two restriction enzyme cleavage sites in each of two primers used to amplify gene-specific fragments from cDNA. This design minimizes the number of PCR primers and results in the placement of unique restriction enzyme recognition sites to allow for flexibility in future manipulations of the plasmid, *e*.*g*., moving the inverted repeat target sequence to a different vector (Chrom database). These vectors are based on pCAMBIA binary vectors, a set of plasmids developed by the Center for Application of Molecular Biology to International Agriculture (CAMBIA).

The pHELLSGATE, high-throughput gene silencing vector and a high throughput tobacco rattle virus (TRV) based VIGS vector are binary vectors developed by Invitrogen are used for expression of GUS and GFP proteins. These vectors are base on Gateway recombinationbased technology, which replaced the conventional cloning strategy. It is based on the phage lambda system of recombination. It enables segments of DNA to be transferred between different vectors while orientation and reading frame are maintained. It can also be used for transfer of PCR products. It saves valuable time, because once the DNA has been cloned into a Gateway vector, it can be used as many genome function analysis systems as is required. In this way, the use of vectors in the process of plant functional genomics has been made much easier, while the process has also been made faster. This allows for higher throughput analysis to occur [88].

#### **3.2. RNAi and functional genomic studies**

An important application of RNAi for functional genomics studies is to generate lines that are deficient for the activity of a subset of genes and then test the knockdown lines for a specific phenotype. The assessing of a specific phenotype requires the presence of a specific allele of marker genes and several generation of crosses are necessary for selecting a specific mutant allele for specific genotype. RNAi technology for functional genomics has advantage that a specific gene can be silenced if the target sequence is better chosen. However, since RNAi is a homology-dependent process a carefully selection of a unique or conserved region of the target gene ensures that a specific member of a multiples gene family can be silenced. For example, RNAi can down-regulate specific target sequences when 3'UTR region is used as a trigger sequence [89-90]. It has been also shown that RNAi facilitates the generation of dominant loss-of-function mutation in polyploidy plants, even with short dsRNAs of a 37 nucleotide long [91].

Nowadays, numerous projects are lunched to produce siRNAs that will silence essential genes in insects, nematodes and pathogens using an approach called hdRNAi (hostdelivered RNAi) [92-94] based on the partial sequences similarities between plant and animal genes. There is also a limitation of this approach because, some unexpected genes can be silenced with consequences on the organisms itself but also environment.

RNAi has been also used for the improvement of nutritional value of some important crops. For example, to decrease the levels of natural toxins in food plants a stable, heritable and distinct siRNA against the toxin could be used. Cottonseeds are rich in dietary proteins but unpalatable by humans as they contain a natural toxic terpenoid item, called gossypol. RNAi mechanism has been used to minimize the levels of delta-cadinene synthase, an enzyme crucial for the production of gossypol [95].

RNAi technology has been also applied to barley for developing varieties resistant to BYDV (barley yellow dwarf virus) [96]. In rice, RNAi has been used to reduce the level of glutein and produce rice varieties with low-glutein content [97]. Soybeans can be engineered to produce oil with low levels of polyunsaturated fatty acids through a reduction of FAD2, a fatty acyl Δ12 desaturase. This enzyme converts the monounsaturated fatty acid oleic acid (18:1Δ9) to linoleic acid (18:2Δ9, Δ12), which can be subsequently desaturated to α-linolenic acid (18:3Δ9, Δ12, Δ15) by FAD3 [98]. The reduced polyunsaturated fatty acid levels from >65% of the total oil content in normal soybean oil to less than 5% was observed [99]. In an attempt to specifically target FAD2-1, and not related family members, the soybean FAD2-1A intron was tested as an RNAi trigger, resulting in a reduction in polyunsaturated fatty acids in the seeds to about 20% [100]. This result was surprising, given that intron sequences are removed from precursor mRNAs (pre-mRNAs) by splicing in the nucleus and spatially separated from the cytoplasm where mature mRNAs are presumed to be targeted by the PTGS machinery [101].

There are also some limitations of using RNAi in functional genomics studies. Unlike the insertional mutagenesis, for the use of RNAi the exact sequence of the target gene is required. Secondly, the methods to delivery RNAi are very important, some species are easily transformable and some not. Nonetheless, further improvement of the delivery methods and vectors that can be used safely and reliably are needed. There have also been some reports that revealed the difficulty to detect mutants with subtle changes in gene expression. However, in plants, numerous marker genes are being developed that will indicate if a change in gene expression occurs [102].

#### **3.3. RNAi and viral infections**

78 Functional Genomics

knockdown of *Arabidopsis* genes.

analysis to occur [88].

Biology to International Agriculture (CAMBIA).

**3.2. RNAi and functional genomic studies**

Currently, several vectors used for RNAi silencing that make use of *Agrobacterium* mediated delivery or artificially introduced dsRNA and/or VIGS into plants has been reported. For example, in 2007, an *Arabidopsis* genomic RNAi knock-out line analysis consortium was lunched out (AGRIKOLA) which is using the PCR products to generate gene-specific RNAi constructs for each *Arabidopsis* gene used in large scale gene silencing studies [86-87]; other consortium called CATMA (Complete *Arabidopsis* Transcriptome MicroArray), is generating gene sequence tags (GSTs) representing each *Arabidopsis* gene, designed so that they will hybridize on *Arabidopsis* cDNA microarrays in a gene-specific manner; the *Medicago truncatula* RNAi database (https://mtrnai.msi.umn.edu/) is a NSF-funded project planning to silence 1500 genes involved in symbiosis in this model legume; amiRNAi Central (http://www.agrikola.org) a NSF project funded to provide a comprehensive resource for

Moreover, a set of binary vectors, called ChromDB's RNAi vectors, were designed for producing dominant negative RNAi mutants using a target sequence cloning strategy that is based on the inclusion of two restriction enzyme cleavage sites in each of two primers used to amplify gene-specific fragments from cDNA. This design minimizes the number of PCR primers and results in the placement of unique restriction enzyme recognition sites to allow for flexibility in future manipulations of the plasmid, *e*.*g*., moving the inverted repeat target sequence to a different vector (Chrom database). These vectors are based on pCAMBIA binary vectors, a set of plasmids developed by the Center for Application of Molecular

The pHELLSGATE, high-throughput gene silencing vector and a high throughput tobacco rattle virus (TRV) based VIGS vector are binary vectors developed by Invitrogen are used for expression of GUS and GFP proteins. These vectors are base on Gateway recombinationbased technology, which replaced the conventional cloning strategy. It is based on the phage lambda system of recombination. It enables segments of DNA to be transferred between different vectors while orientation and reading frame are maintained. It can also be used for transfer of PCR products. It saves valuable time, because once the DNA has been cloned into a Gateway vector, it can be used as many genome function analysis systems as is required. In this way, the use of vectors in the process of plant functional genomics has been made much easier, while the process has also been made faster. This allows for higher throughput

An important application of RNAi for functional genomics studies is to generate lines that are deficient for the activity of a subset of genes and then test the knockdown lines for a specific phenotype. The assessing of a specific phenotype requires the presence of a specific allele of marker genes and several generation of crosses are necessary for selecting a specific mutant allele for specific genotype. RNAi technology for functional genomics has advantage that a specific gene can be silenced if the target sequence is better chosen. However, since RNAi is a homology-dependent process a carefully selection of a unique or conserved

RNA silencing in plants prevents viral accumulation and accordingly, viruses have evolved several strategies to counteract the defense mechanism. A viral protein, HC-Pro (helper

component proteinase) was shown to mediate one class of viral synergism disease [103] and expression of this protein in transgenic plants allows the accumulation of heterologous viruses beyond the normal level suggesting that HC-Pro blocked the target plant defence mechanism [104]. There are several methods known to identify viral suppressor proteins, such as transient expression assay, the reversal of silencing assay and stable expression assay.

RNAi Towards Functional Genomics Studies 81

whose expression is know to be up-regulated in a disease, given an appropriate tissue culture model of that disease. The libraries of RNAi reagents can be used in one of two ways. One is in a high throughput manner, in which each gene in the genome is knocked down one at a time. The other approach is to use large pools of RNA interference viral vectors and apply a selective pressure that only cells with the desired change in behavior can survive [112]. Rapid progress in the application of RNAi to mammalian cells, including neurons, muscle cells, offers new approaches to drug target identification. Advances in targeted delivery of RNAi-inducing molecules has raised the possibility of using RNAi

Considering the gene-specific features of RNAi, it is conceivable that this method it will be very useful for therapeutic applications. Direct transfection of siRNAs into cells, creating an expression construct in which a promoter drives the production of both the sense and antisense siRNAs which then hybridize in the cell to produce the double stranded siRNA and using viral vectors to infect cells with an expression construct are the methods used

Nonetheless, this hypothesis is based on the assumption that the effect of exogenous siRNA applications will remain gene specific and do not show nonspecific side effects relating to mismatches off-target hybridization or protein binding to nucleic acids. For example, several research groups have explored the use of RNAi to limit infection by viruses in cultured cells. There is a huge potential for using RNAi for the treatment of viral diseases such as those

RNAi strategy includes multiple targets to neutralize HIV. For example, directed siRNAs against several regions of the HIV-1 genome, including the viral long terminal repeat (LTR) and the accessory genes, *vif* and *nef* [113-115]. Using Magi cells (CD4-positive HeLa cells) as a model system, they demonstrated a sequence specific reduction of >95% in viral infection after cotransfection of siRNAs with an HIV-1 proviral DNA. When the same assay was done in primary peripheral blood lymphocytes, which are natural targets for HIV-1, the frequency of infected cells was also substantially reduced. These could be targets that block entry into the cell and disrupts the virus reproduction cycle inside the cells. This technology will help researchers dissect the biology of HIV infection and design drugs based on this molecular information [116]. Researchers from Hope Cancer Center in Duarte have developed a DNA-based delivery system in which human cells are generated to produce

The delivery of siRNA to HIV-infected T lymphocytes, monocytes and macrophages is a challenge. As synthetic siRNAs do not persist for long periods in cells, they would have to be delivered repeatedly for years to treat the infection. Systemic delivery of siRNAs to lymphocytes is not feasible owning to the huge number of these cells. Therefore, the preferred method is to isolate T cells from patients. In clinical trial T cells from HIV-infected individuals are transduced ex vivo with a lentiviral vector that encodes an anti-HIV antisense RNA, and then reinfused into patients [118]. In other study, it was reported that

caused by the human immunodeficiency virus (HIV) and the hepatitis C virus.

siRNA against REV protein, which is important in causing AIDS [117].

directly as a therapy for a variety of human genetic disorders.

**4.2. RNAi and therapy**

nowadays for RNAi-based therapy.

A well known used method to study the transient expression is co-infiltration method using *Agrobacterium* strains, one strain used for inducing of RNA silencing of a reporter gene such as GFP (green fluorescent protein) and one strain that will express the candidate suppressor gene. Both strains will be infiltrated in a plant tissues such tobacco leaves, which are suited for production of a higher amount of protein in response to agro-infiltration. However, if the local silencing is triggered three days after infiltration the effect can be evaluated under UV light. If the candidate suppressor expressed from the co-infiltrated *Agrobacterium* interferes with RNA silencing, the tissue will remain bright green and in case not, the tissue will turn red [105]. In the case of reversal approach the candidates that may suppress silencing are identified. Several studies have shown that the viral suppressor proteins play an important role in this defense mechanism [106]. For two suppressor proteins, p21 encoded by beet western yellow virus [107] and p19 encoded by the tomato bushy stunt virus (TBSV) group [108] the molecular mechanism was identified.

Stable expression assay approach, a stable RNAi line expressing a suppressor candidate is crossed with several lines silenced for a repressor gene [109-110]. This method is also advantageous because provide information about the molecular mechanisms of the suppression and is also suited to investigate the role of suppression in systemic silencing using grafting [111].

However, the findings that certain viral proteins suppress RNA silencing open a new tool for biotechnologies applications. With silencing under control, many transgenic plants can be generated to produce desired plant traits or very higher level of expression to use the plant as a factor for producing pharmaceutical compounds, vaccines and other gene-products.

#### **4. Application of RNAi in animal systems**

#### **4.1. RNAi and medicine**

The ability to trigger RNAi in mammals was first demonstrated by microinjection of long dsRNA into mouse oocytes and one-cell stage embryos [19]. In this case was demonstrated that the antiviral interferon response to long dsRNAs is not yet functional in early mouse embryos. It was discovered rather quickly that chemically synthesized siRNAs could trigger sequence-specific silencing in cultured mammalian cells without inducing the interferon response [21]. Starting from this important breakthrough, RNAi has emerged as a powerful experimental tool for analyzing mammalian systems.

The ability of RNAi to determine ablation of gene expression has open up the possibility of using collections of siRNAs to analyze the role of hundreds or thousands of different genes whose expression is know to be up-regulated in a disease, given an appropriate tissue culture model of that disease. The libraries of RNAi reagents can be used in one of two ways. One is in a high throughput manner, in which each gene in the genome is knocked down one at a time. The other approach is to use large pools of RNA interference viral vectors and apply a selective pressure that only cells with the desired change in behavior can survive [112]. Rapid progress in the application of RNAi to mammalian cells, including neurons, muscle cells, offers new approaches to drug target identification. Advances in targeted delivery of RNAi-inducing molecules has raised the possibility of using RNAi directly as a therapy for a variety of human genetic disorders.

#### **4.2. RNAi and therapy**

80 Functional Genomics

assay.

[108] the molecular mechanism was identified.

**4. Application of RNAi in animal systems**

experimental tool for analyzing mammalian systems.

using grafting [111].

**4.1. RNAi and medicine**

component proteinase) was shown to mediate one class of viral synergism disease [103] and expression of this protein in transgenic plants allows the accumulation of heterologous viruses beyond the normal level suggesting that HC-Pro blocked the target plant defence mechanism [104]. There are several methods known to identify viral suppressor proteins, such as transient expression assay, the reversal of silencing assay and stable expression

A well known used method to study the transient expression is co-infiltration method using *Agrobacterium* strains, one strain used for inducing of RNA silencing of a reporter gene such as GFP (green fluorescent protein) and one strain that will express the candidate suppressor gene. Both strains will be infiltrated in a plant tissues such tobacco leaves, which are suited for production of a higher amount of protein in response to agro-infiltration. However, if the local silencing is triggered three days after infiltration the effect can be evaluated under UV light. If the candidate suppressor expressed from the co-infiltrated *Agrobacterium* interferes with RNA silencing, the tissue will remain bright green and in case not, the tissue will turn red [105]. In the case of reversal approach the candidates that may suppress silencing are identified. Several studies have shown that the viral suppressor proteins play an important role in this defense mechanism [106]. For two suppressor proteins, p21 encoded by beet western yellow virus [107] and p19 encoded by the tomato bushy stunt virus (TBSV) group

Stable expression assay approach, a stable RNAi line expressing a suppressor candidate is crossed with several lines silenced for a repressor gene [109-110]. This method is also advantageous because provide information about the molecular mechanisms of the suppression and is also suited to investigate the role of suppression in systemic silencing

However, the findings that certain viral proteins suppress RNA silencing open a new tool for biotechnologies applications. With silencing under control, many transgenic plants can be generated to produce desired plant traits or very higher level of expression to use the plant as

The ability to trigger RNAi in mammals was first demonstrated by microinjection of long dsRNA into mouse oocytes and one-cell stage embryos [19]. In this case was demonstrated that the antiviral interferon response to long dsRNAs is not yet functional in early mouse embryos. It was discovered rather quickly that chemically synthesized siRNAs could trigger sequence-specific silencing in cultured mammalian cells without inducing the interferon response [21]. Starting from this important breakthrough, RNAi has emerged as a powerful

The ability of RNAi to determine ablation of gene expression has open up the possibility of using collections of siRNAs to analyze the role of hundreds or thousands of different genes

a factor for producing pharmaceutical compounds, vaccines and other gene-products.

Considering the gene-specific features of RNAi, it is conceivable that this method it will be very useful for therapeutic applications. Direct transfection of siRNAs into cells, creating an expression construct in which a promoter drives the production of both the sense and antisense siRNAs which then hybridize in the cell to produce the double stranded siRNA and using viral vectors to infect cells with an expression construct are the methods used nowadays for RNAi-based therapy.

Nonetheless, this hypothesis is based on the assumption that the effect of exogenous siRNA applications will remain gene specific and do not show nonspecific side effects relating to mismatches off-target hybridization or protein binding to nucleic acids. For example, several research groups have explored the use of RNAi to limit infection by viruses in cultured cells. There is a huge potential for using RNAi for the treatment of viral diseases such as those caused by the human immunodeficiency virus (HIV) and the hepatitis C virus.

RNAi strategy includes multiple targets to neutralize HIV. For example, directed siRNAs against several regions of the HIV-1 genome, including the viral long terminal repeat (LTR) and the accessory genes, *vif* and *nef* [113-115]. Using Magi cells (CD4-positive HeLa cells) as a model system, they demonstrated a sequence specific reduction of >95% in viral infection after cotransfection of siRNAs with an HIV-1 proviral DNA. When the same assay was done in primary peripheral blood lymphocytes, which are natural targets for HIV-1, the frequency of infected cells was also substantially reduced. These could be targets that block entry into the cell and disrupts the virus reproduction cycle inside the cells. This technology will help researchers dissect the biology of HIV infection and design drugs based on this molecular information [116]. Researchers from Hope Cancer Center in Duarte have developed a DNA-based delivery system in which human cells are generated to produce siRNA against REV protein, which is important in causing AIDS [117].

The delivery of siRNA to HIV-infected T lymphocytes, monocytes and macrophages is a challenge. As synthetic siRNAs do not persist for long periods in cells, they would have to be delivered repeatedly for years to treat the infection. Systemic delivery of siRNAs to lymphocytes is not feasible owning to the huge number of these cells. Therefore, the preferred method is to isolate T cells from patients. In clinical trial T cells from HIV-infected individuals are transduced ex vivo with a lentiviral vector that encodes an anti-HIV antisense RNA, and then reinfused into patients [118]. In other study, it was reported that

the GFP siRNA induced gene silencing of transient or stably expressed GFP mRNA was highly specific in the human embryonic kidney (HEK) 293 cell background [119]. Further study, in human non-cell lung carcinoma cell line H1299 have shown that specific siRNAs corresponding to *akt1*, *rb1*, and *plk1* could be used as highly specific tools for targeted gene knockdown and can be used in high-throughput approaches and drug target validation.

RNAi Towards Functional Genomics Studies 83

Defects in miRNA expression could cause the development of cancer associated with impaired formation of oncoproteins or tumor supressors regulated by these miRNAs. One of the first identified miRNAs, let-7 found in *C. elegans* as well as in humans, is a tumor suppressor decreasing formation of the oncoproteins Ras and HMGA2 (High Mobility Group protein A2). In patients with lung cancer was a inverse correlation between expression of let-7 and Ras/HMGA2 [125]. miR-155 is an exemple of miRNA exhibiting oncogenic properties. This miRNA is required for B- and T- cells functioning [126]. Increased expression of miR-155 was observed in paediatric Burkitt's lymphoma, diffuse large cell lymphoma, Hodgkin's disease, lung, breast and pancreatic cancers [124]. In some tumor cells miR-21 and mi-R 24 act as oncogenes, and in others they act as tumor supressors. In HeLa cells, inhibition of miR-21 or miR-24 activity by modified anti-miR oligonucleotides accelerated proliferation [127]. Analysis of bioinformatic predictions of putative targets

suggests that proto- and anti-oncogenic activity is typical for various miRNA [128].

of tumors [130].

Defects in miRNA expression associated with carcinogens are caused not only by the chromosomal rearrangements, but also due to impairments in the machinery responsible for miRNA formation and processing. Inhibition of expression of ribonucleases Dicer and Drosha by complementary siRNA caused acceleration of growth of lung adenocarcinoma cells in murine model [129]. Tumor transformation may be also determined by primary impairments in regulation of expression of a single miRNA, which is then accompanied by imbalance in the entire miRNA network [124]. Impairments in miRNA functioning seen in cancerogenesis can be used for detection of miRNA expression for diagnostics of tumor origin. Each type of cancer is characterized by a certain pattern of miRNA expression [124]. Moreover, the evaluation of miRNA profiles can be used for prognosis of the development

The importance of epigenomic modifications of chromatin structure for the development of tumors has been recognized [13]. Methylation of cytosine in DNA followed by formation of 5 methylCytozine occurs in dinucleotide sequence CpG. Methylation can cause suppression of transcription by proteins recognizing methylated CpG attraction. Excesive methylation of CpG island of miRNA genes has been found in cancer cells [131]. The authors attribute the effect of this methylation to suppression of tumor suppressor genes represented by miRNA genes. Processes of DNA methylation may be closely associated with modification of chromatin histones. Some histone modifications like acetylation, methylation and phosphorylation of specific aminoacides residues are involved in gene expression. Impairments of histone modifications in tumor cells have been found in many studies [132]. Processes of epigenomic silencing may involve another type of non-coding RNAs, short siRNAs. This observation demonstrates the existence of nuclear RNA-interference, which is based on suppression of mRNA translation. Some experimental data suggest that siRNA plays a certain role in gene silencing at the level of chromatin in human cancer cells. There are some examples of involvement of short RNAs in epigenomic silencing, which is coupled to DNA methylation, histone modification of target genes, and attraction of the heterochromatin HP1 protein to them. All these chromatin modifications typically occur in the cancer epigenome [124]. The role of RNAi is recognized not only in silencing of proto-oncogenes or

RNAi is also utilized as an antiviral therapy against diseases caused by herpes simplex virus type 2, hepatits A and hepatits B. Early RNAi studies noted that RNA silencing was prominent in the liver, which made this organ an attractive target for therapeutic approaches. Vaccine against HBV is used only for prevention and there is no vaccine for HCV. Mc Caffrey and his co-workers (2003) [120] demonstrated that a significant knockdown of the HBV core antigen in liver hepatocytes could be achieved by the siRNA, providing an important proof of principle for future antiviral applications of RNAi. They developed a transient model of HBV infection in which a plasmid containing approximately 1-3 copies of the HBV genome (pTHBV2) was introduced into the livers of mice by hydrodynamic transfection. This results in production of all four families of viral mRNAs, including the pregenomic RNA. The pregenomic RNA is the template for the viral reverse transcriptase, which replicates new viral DNA. All four viral proteins are also made. Transient viral replication occured in the mouse liver for about 1 week. In addition, RNAi has achieved regression of clinical traits in neurodegenerative disease model [121] but its potential for use in pharmaceutical target validation and as a therapeutic tool is still ongoing.

The ability to induce RNAi across mucosal surfaces was also investigated as a means for treating de sexually transmitted disorders [122]. siRNA targeting tumor necrosis factor alpha was injected into the joints of mice with collagen induced arthritis (CIA) and the development of arthritis was scored by assessing the inflammation of joints in the mouse paw, and the mice with CIA, joint inflammation was successfully inhibited [123]. Antiviral RNAi therapeutics have already entered human clinical trials and will hopefully prove to be safe and efcacious.

#### **4.3. RNAi and cancer**

The discovery of RNAi led to the realization that the RNAi machinery is also involved in normal gene regulation through the action of a class of small RNAs known as microRNAs. There is experimental evidence that miRNAs regulate cell division, differentiation, cell fate decisions, development, oncogenesis, apoptosis, and many other processes [35]. miRNA levels are also dramatically shifted in various cancers, and miRNAs can act as oncogenes [35]. It is now clear that miRNAs represent a gene regulatory network of enormous signicance. The expression profile of miRNA is highly specific for a particular type of tissue and cell stage of cell differentiation [124]. Impaired miRNA functioning, which occurs during tumor transformation, can be evaluated as a consequence rather than the cause of loss of cell identity. However, detection of chromosomal rearrangement like deletions, local amplifications and chromosomal breackpoints in the region of miRNA genes (causing impairments in miRNA expression during cancerogenesis) is a good demonstration of direct role of miRNA in these processes.

Defects in miRNA expression could cause the development of cancer associated with impaired formation of oncoproteins or tumor supressors regulated by these miRNAs. One of the first identified miRNAs, let-7 found in *C. elegans* as well as in humans, is a tumor suppressor decreasing formation of the oncoproteins Ras and HMGA2 (High Mobility Group protein A2). In patients with lung cancer was a inverse correlation between expression of let-7 and Ras/HMGA2 [125]. miR-155 is an exemple of miRNA exhibiting oncogenic properties. This miRNA is required for B- and T- cells functioning [126]. Increased expression of miR-155 was observed in paediatric Burkitt's lymphoma, diffuse large cell lymphoma, Hodgkin's disease, lung, breast and pancreatic cancers [124]. In some tumor cells miR-21 and mi-R 24 act as oncogenes, and in others they act as tumor supressors. In HeLa cells, inhibition of miR-21 or miR-24 activity by modified anti-miR oligonucleotides accelerated proliferation [127]. Analysis of bioinformatic predictions of putative targets suggests that proto- and anti-oncogenic activity is typical for various miRNA [128].

82 Functional Genomics

be safe and efcacious.

**4.3. RNAi and cancer**

role of miRNA in these processes.

the GFP siRNA induced gene silencing of transient or stably expressed GFP mRNA was highly specific in the human embryonic kidney (HEK) 293 cell background [119]. Further study, in human non-cell lung carcinoma cell line H1299 have shown that specific siRNAs corresponding to *akt1*, *rb1*, and *plk1* could be used as highly specific tools for targeted gene knockdown and can be used in high-throughput approaches and drug target validation.

RNAi is also utilized as an antiviral therapy against diseases caused by herpes simplex virus type 2, hepatits A and hepatits B. Early RNAi studies noted that RNA silencing was prominent in the liver, which made this organ an attractive target for therapeutic approaches. Vaccine against HBV is used only for prevention and there is no vaccine for HCV. Mc Caffrey and his co-workers (2003) [120] demonstrated that a significant knockdown of the HBV core antigen in liver hepatocytes could be achieved by the siRNA, providing an important proof of principle for future antiviral applications of RNAi. They developed a transient model of HBV infection in which a plasmid containing approximately 1-3 copies of the HBV genome (pTHBV2) was introduced into the livers of mice by hydrodynamic transfection. This results in production of all four families of viral mRNAs, including the pregenomic RNA. The pregenomic RNA is the template for the viral reverse transcriptase, which replicates new viral DNA. All four viral proteins are also made. Transient viral replication occured in the mouse liver for about 1 week. In addition, RNAi has achieved regression of clinical traits in neurodegenerative disease model [121] but its potential for use in pharmaceutical target validation and as a therapeutic tool is still ongoing. The ability to induce RNAi across mucosal surfaces was also investigated as a means for treating de sexually transmitted disorders [122]. siRNA targeting tumor necrosis factor alpha was injected into the joints of mice with collagen induced arthritis (CIA) and the development of arthritis was scored by assessing the inflammation of joints in the mouse paw, and the mice with CIA, joint inflammation was successfully inhibited [123]. Antiviral RNAi therapeutics have already entered human clinical trials and will hopefully prove to

The discovery of RNAi led to the realization that the RNAi machinery is also involved in normal gene regulation through the action of a class of small RNAs known as microRNAs. There is experimental evidence that miRNAs regulate cell division, differentiation, cell fate decisions, development, oncogenesis, apoptosis, and many other processes [35]. miRNA levels are also dramatically shifted in various cancers, and miRNAs can act as oncogenes [35]. It is now clear that miRNAs represent a gene regulatory network of enormous signicance. The expression profile of miRNA is highly specific for a particular type of tissue and cell stage of cell differentiation [124]. Impaired miRNA functioning, which occurs during tumor transformation, can be evaluated as a consequence rather than the cause of loss of cell identity. However, detection of chromosomal rearrangement like deletions, local amplifications and chromosomal breackpoints in the region of miRNA genes (causing impairments in miRNA expression during cancerogenesis) is a good demonstration of direct Defects in miRNA expression associated with carcinogens are caused not only by the chromosomal rearrangements, but also due to impairments in the machinery responsible for miRNA formation and processing. Inhibition of expression of ribonucleases Dicer and Drosha by complementary siRNA caused acceleration of growth of lung adenocarcinoma cells in murine model [129]. Tumor transformation may be also determined by primary impairments in regulation of expression of a single miRNA, which is then accompanied by imbalance in the entire miRNA network [124]. Impairments in miRNA functioning seen in cancerogenesis can be used for detection of miRNA expression for diagnostics of tumor origin. Each type of cancer is characterized by a certain pattern of miRNA expression [124]. Moreover, the evaluation of miRNA profiles can be used for prognosis of the development of tumors [130].

The importance of epigenomic modifications of chromatin structure for the development of tumors has been recognized [13]. Methylation of cytosine in DNA followed by formation of 5 methylCytozine occurs in dinucleotide sequence CpG. Methylation can cause suppression of transcription by proteins recognizing methylated CpG attraction. Excesive methylation of CpG island of miRNA genes has been found in cancer cells [131]. The authors attribute the effect of this methylation to suppression of tumor suppressor genes represented by miRNA genes. Processes of DNA methylation may be closely associated with modification of chromatin histones. Some histone modifications like acetylation, methylation and phosphorylation of specific aminoacides residues are involved in gene expression. Impairments of histone modifications in tumor cells have been found in many studies [132]. Processes of epigenomic silencing may involve another type of non-coding RNAs, short siRNAs. This observation demonstrates the existence of nuclear RNA-interference, which is based on suppression of mRNA translation. Some experimental data suggest that siRNA plays a certain role in gene silencing at the level of chromatin in human cancer cells. There are some examples of involvement of short RNAs in epigenomic silencing, which is coupled to DNA methylation, histone modification of target genes, and attraction of the heterochromatin HP1 protein to them. All these chromatin modifications typically occur in the cancer epigenome [124]. The role of RNAi is recognized not only in silencing of proto-oncogenes or tumor suppressors, but also in maintenance of heterochromatin structure of centromeric region in mammalian cells [133].

RNAi Towards Functional Genomics Studies 85

The Nobel Prize-winning research on RNA inhibition might lead to new treatments for patients with this disease due to dominant mutations in the superoxide dismutase gene. Reduction in the level of the superoxide dismutase enzyme coded for by the mutant gene has been studied in animal models [143]. Working with laboratory mice as an experimental model system for the human disease ALS*,* Miller and coworkers showed that loss of muscle function could be slowed using RNA interference [144]. This result was obtained by using a virus to induce RNA interference in neurons. These results from laboratory experiments suggest that if RNA-induced inhibition of mutant superoxide dismutase can be induced in the correct cells of the brain and spinal cord, it might be possible to slow progression of *Lou*

A high number of pharmaceutical and biotechnology companies have declared an interest in or have an active drug development program already underway in RNAi-based therapeutics to silence disease associated genes. This web (www.rnaiweb.com) collects together the latest research covering the development of RNAi based tools for drug target and gene function analysis. These include Sirna Therapeutics (Colorado) for macular degeneration; Avocel (Sunnyvale, California) for hepatits C; Alnylam Pharmaceuticals (Cambridge) for Parkinson's disease; CytRx (Los Angeles, California) for obesity, type II diabetes and ALS etc. But the major challenge in turning RNAi into an effective therapeutic strategy is the delivery of the RNAi agents, whether they are synthetic short double stranded RNAs or viral vectors directing production of double stranded RNA. During diseases, changes in the pattern of microRNAs will occur with some being indicative of treatment outcome and disease progression. More exciting then diagnostic value is the evidence that directly involves miRNAs in a number of diseases (cancer, imprinting impairments etc). In this context there is an increased interest in manipulations miRNAs for therapeutic purposes. In the loos of miRNA function, one approch is to mimic miRNA activity by introducing microRNA "mimics"with the same genetic information as the natural miRNA. For exemple, by adding more of a microRNA named let-t, it has been

Another complementary approach to using miRNAs for therapy is to inhibit the activity of disease-associated miRNAs. This can be achieved by employing antisense oligonucleotides that, based on sequence complementarity, will bind to inactivate miRNA function. Esau end colleagues demonstrated that the inhibition of miRNA may be a potential therapeutic approach to the treatment of disease [146]. They inhibited miR-122 expression with antagomirs, which resulted in reduced plasma cholesterol levels and a decrease in hepatic fatty aceid and cholesterol synthesis rates in normal mice and in diet-induced obese mice.

RNA interference has much promise in laboratory. In principle, RNAi might be used to treat any disease that is linked to expression of an identified gene [112]. The most important challenge in turning RNA interference into an effective therapeutic strategy is the delivery of the RNA interference agents. Given sufficient research into delivery methods, some of

Targeting miR-122 with antagomirs resulted in inhibition of disease development.

these diseases will probably be treated effectively by RNAi based therapeutics.

*Gehrig's disease* in humans.

possible to halt cancer cells from further multiplying [145].

#### **4.4. Challenges for RNAi as a tool for diseases inverstigations**

One of the advantages of RNAi over gene knockout is the ability to restrict gene knockdown to specific tissues or even cell types. This is important when a disease is a result of a mutation in an essential gene. The versatility of the technique has led to many applications. RNAi can be used in drug target validation and RNAi can target specific spliced exons, enabling the investigation of the functional roles of alternatively spliced forms of a gene [134]. An important opportunity is the use of RNAi in identification of all candidate genes involved in certain physiological processes using genome-wide RNAi screening [135].

RNAi can be applied to genetic model organisms such *Drosophila*, *C. elegans* and mouse in order to investigate and/or to treat some human disorders. Several models of human neural and neuromuscular disorders are available in this three experimental models. *C. elegans* is model for RNAi knockdown of genes to mimic loss of in order to elucidate the mechanisms of a number of muscle wasting diseases like: Duchenne muscular dystrophy [136], X-linked form of Emery-Dreifuss muscular distrophy [137], spinal muscular atrophy [137], fragile Xsyndrome [138], Alzheimer's disease [139]. RNAi has been used in combination with overexpression studies to study the role of Parkin, an E3 ubiquitin ligase, in dopamine neuron degeneration in *Drosophila* in order to investigate the molecular mechanisms underlying Parkinson's disease. Overexpression of Parkin was shown to degrade its substrate (Pael-R) and suppress its toxicity, whereas interfering with endigenous Parkin promoted substrate accumulation and augmented its neurotoxicity [140].

Other experiment uses *Drosophila* in order to investigate human neurodegenerative disorders that are caused by expansion of CAG trinucleotide repeat. RNAi has been performed on two such diseases: Huntington's disease and spinobulbar muscular atrophy. *Drosophila* S2 cells were engineered to express a portion of human ar gene with CAG tracs of 26, 43 or 106 repeats tagged by green fluorescent protein (GFP) [141]. Cells carrying CAG repeats of 43 and 106 developed GFP aggregates. Using RNAi directed against AR protein, a loos of AR-GFP aggregates by 80% in co-transfected S2 cells has been observed. Therefore, RNAi could have considerable therapeutic potential in neurodegenerative disorders [134]. Murine model was used to investigate the potential of RNAi as therapeutic tool in neurodegenerative disorders. Spinocerebellar ataxia type 1 (SCA1) has been successfully suppressed by RNAi in mouse model of this disease [121].

RNAi might also allow future treatments of human disease such as Lou Gehrig's disease (amyotrophic lateral sclerosis, ALS), a genetically dominant inherited disease. This pattern of inheritance allowed identification of a specific gene that is linked to the death of Betz cells in some families, the gene for an enzyme called superoxide dismutase. Superoxide dismutase can protect cells from molecular damage caused by free radicals of oxygen. Mutant forms of superoxide dismutase can lead to cell death [142].

The Nobel Prize-winning research on RNA inhibition might lead to new treatments for patients with this disease due to dominant mutations in the superoxide dismutase gene. Reduction in the level of the superoxide dismutase enzyme coded for by the mutant gene has been studied in animal models [143]. Working with laboratory mice as an experimental model system for the human disease ALS*,* Miller and coworkers showed that loss of muscle function could be slowed using RNA interference [144]. This result was obtained by using a virus to induce RNA interference in neurons. These results from laboratory experiments suggest that if RNA-induced inhibition of mutant superoxide dismutase can be induced in the correct cells of the brain and spinal cord, it might be possible to slow progression of *Lou Gehrig's disease* in humans.

84 Functional Genomics

region in mammalian cells [133].

in mouse model of this disease [121].

tumor suppressors, but also in maintenance of heterochromatin structure of centromeric

One of the advantages of RNAi over gene knockout is the ability to restrict gene knockdown to specific tissues or even cell types. This is important when a disease is a result of a mutation in an essential gene. The versatility of the technique has led to many applications. RNAi can be used in drug target validation and RNAi can target specific spliced exons, enabling the investigation of the functional roles of alternatively spliced forms of a gene [134]. An important opportunity is the use of RNAi in identification of all candidate genes involved in certain physiological processes using genome-wide RNAi screening [135].

RNAi can be applied to genetic model organisms such *Drosophila*, *C. elegans* and mouse in order to investigate and/or to treat some human disorders. Several models of human neural and neuromuscular disorders are available in this three experimental models. *C. elegans* is model for RNAi knockdown of genes to mimic loss of in order to elucidate the mechanisms of a number of muscle wasting diseases like: Duchenne muscular dystrophy [136], X-linked form of Emery-Dreifuss muscular distrophy [137], spinal muscular atrophy [137], fragile Xsyndrome [138], Alzheimer's disease [139]. RNAi has been used in combination with overexpression studies to study the role of Parkin, an E3 ubiquitin ligase, in dopamine neuron degeneration in *Drosophila* in order to investigate the molecular mechanisms underlying Parkinson's disease. Overexpression of Parkin was shown to degrade its substrate (Pael-R) and suppress its toxicity, whereas interfering with endigenous Parkin

Other experiment uses *Drosophila* in order to investigate human neurodegenerative disorders that are caused by expansion of CAG trinucleotide repeat. RNAi has been performed on two such diseases: Huntington's disease and spinobulbar muscular atrophy. *Drosophila* S2 cells were engineered to express a portion of human ar gene with CAG tracs of 26, 43 or 106 repeats tagged by green fluorescent protein (GFP) [141]. Cells carrying CAG repeats of 43 and 106 developed GFP aggregates. Using RNAi directed against AR protein, a loos of AR-GFP aggregates by 80% in co-transfected S2 cells has been observed. Therefore, RNAi could have considerable therapeutic potential in neurodegenerative disorders [134]. Murine model was used to investigate the potential of RNAi as therapeutic tool in neurodegenerative disorders. Spinocerebellar ataxia type 1 (SCA1) has been successfully suppressed by RNAi

RNAi might also allow future treatments of human disease such as Lou Gehrig's disease (amyotrophic lateral sclerosis, ALS), a genetically dominant inherited disease. This pattern of inheritance allowed identification of a specific gene that is linked to the death of Betz cells in some families, the gene for an enzyme called superoxide dismutase. Superoxide dismutase can protect cells from molecular damage caused by free radicals of oxygen.

**4.4. Challenges for RNAi as a tool for diseases inverstigations**

promoted substrate accumulation and augmented its neurotoxicity [140].

Mutant forms of superoxide dismutase can lead to cell death [142].

A high number of pharmaceutical and biotechnology companies have declared an interest in or have an active drug development program already underway in RNAi-based therapeutics to silence disease associated genes. This web (www.rnaiweb.com) collects together the latest research covering the development of RNAi based tools for drug target and gene function analysis. These include Sirna Therapeutics (Colorado) for macular degeneration; Avocel (Sunnyvale, California) for hepatits C; Alnylam Pharmaceuticals (Cambridge) for Parkinson's disease; CytRx (Los Angeles, California) for obesity, type II diabetes and ALS etc. But the major challenge in turning RNAi into an effective therapeutic strategy is the delivery of the RNAi agents, whether they are synthetic short double stranded RNAs or viral vectors directing production of double stranded RNA. During diseases, changes in the pattern of microRNAs will occur with some being indicative of treatment outcome and disease progression. More exciting then diagnostic value is the evidence that directly involves miRNAs in a number of diseases (cancer, imprinting impairments etc). In this context there is an increased interest in manipulations miRNAs for therapeutic purposes. In the loos of miRNA function, one approch is to mimic miRNA activity by introducing microRNA "mimics"with the same genetic information as the natural miRNA. For exemple, by adding more of a microRNA named let-t, it has been possible to halt cancer cells from further multiplying [145].

Another complementary approach to using miRNAs for therapy is to inhibit the activity of disease-associated miRNAs. This can be achieved by employing antisense oligonucleotides that, based on sequence complementarity, will bind to inactivate miRNA function. Esau end colleagues demonstrated that the inhibition of miRNA may be a potential therapeutic approach to the treatment of disease [146]. They inhibited miR-122 expression with antagomirs, which resulted in reduced plasma cholesterol levels and a decrease in hepatic fatty aceid and cholesterol synthesis rates in normal mice and in diet-induced obese mice. Targeting miR-122 with antagomirs resulted in inhibition of disease development.

RNA interference has much promise in laboratory. In principle, RNAi might be used to treat any disease that is linked to expression of an identified gene [112]. The most important challenge in turning RNA interference into an effective therapeutic strategy is the delivery of the RNA interference agents. Given sufficient research into delivery methods, some of these diseases will probably be treated effectively by RNAi based therapeutics.

#### **5. Conclusions**

The study of RNAi has led to a revolution in the understanding of gene expression and the examples in plants, animal and mammalian system as reviewed here, showed the diversity and the potential of RNAi as new approach to replaces the classical genetic technologies and manipulation.

RNAi Towards Functional Genomics Studies 87

[6] van der Vlugt RA, Ruiter RK, Goldbach R (1992) Evidence for sense RNA-mediated protection to PVYN in tobacco plants transformed with the viral coat protein cistron.

[7] Ratcliff FG, MacFarlane SA, Baulcombe DC (1999) Gene silencing without DNA. RNA-

[8] Zamore PD (2002) Ancient pathways programmed by small RNAs. Science 296: 1265-

[10] Fire A, Xu S, Montgomery M, Kostas S, Driver S, Mello C (1998) Potent and specific genetic interference by double-stranded RNA in *Caenorhabditis elegans. Nature* 391: 806-811. [11] Ketting RF, Plasterk RH, (2000) A genetic link between suppression and RNA

[12] Elbashir SM, Lendeckel W, Tuschl T (2001a) RNA interference is mediated by 21- and

[13] Bernstein BE, Meissner A, Lander ES (2007) The mammalian epigenome. *Cell* 128: 669-681. [14] Wargelius A, Ellingsen S, Jose FA (1999) Double stranded RNA induces specific developmental defects in zebrafish embryos. *Biochemical and Biophysical Research*

[15] Lohmann JU, Endl I, Bosch TC (1999) Silencing of developmental genes in

[16] Akashi H, Miyagishi M, Taira K (2001) Suppression of gene expression by RNA interference in cultured plant cells. *Antisense Nucleic Acid Drug Development* 11(6):359-367. [17] Elmayan T, Balzergue S, Beon F, Bourdon V, Daubremet J, Guenet Y, Mourrain P, Palauqui JC, Vernhettes S, Vialle T, Wostrikoff K, Vaucheret H (1998) *Arabidopsis*

[18] Thakur A (2003) RNA interference revolution. *Electronic Journal Of Biotechnology* 6 (1). [19] Wianny F, Zernicka-Goetz M (2000) Specific interference with gene function by double-

[21] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001b) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. *Nature*

[22] Watson JM, Fusaro FM, Wang M, Waterhouse PM (2005) RNA silencing platforms in

[23] Matzke MA, Matzke AJ, Pruss GJ, Vance VB (2001) RNA-based silencing strategy in

[24] Tabara H, Grishok A, Mello CC (1998) RNAi in *C. elegans*: soaking in the genome

[25] Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defence

[26] Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA.

stranded RNA in early mouse development. *Nature Cell Biology* 2: 70-75. [20] Kreutzer R, Limmer S (2000) Kreutzer-Limmer Patent(1999/2000), Germany.

mediated cross-protection between viruses. *Plant Cell* 11: 1207-1216.

[9] Ruvkun G (2001) Glimpses of a tiny RNA world. *Science* 294 (5543): 797-799.

*Plant Molecular Biology* 20: 631-639.

interference in *C. elegans. Nature* 404: 296-298.

Hydra. *Developmental Biology* 214(1): 211-214.

*Communications* 263(1):156-161.

plants. *Febs Letters* 579: 5982-5987.

sequence. *Science* 282 (5388): 430–431.

against viruses. *Nature* 411: 834-842.

*Nature* 431: 343-349.

411: 494-498.

22-nucleotide RNAs. *Genes and Development* 15(2): 188–200.

mutants impaired in cosuppression. *Plant Cell* 10(10):1747-1758.

plants. *Current Opinion In Genetic Development* 11: 221-227.

1269.

Today, the RNAi strategies as a new tool for cheap screen of gene function in organisms for which, a genetic approach was not developed yet. However, after 11 years of extensive research, RNAi has now been demonstrated to function in mammalian cells to alter gene expression and used as a means for genetic discovery as well as a possible strategy for genetic correction and genetic therapy in cancer and other diseases.

Finally, RNAi represent a significant tool for the accomplishment of these goals, and will undoubtedly be used to address many other challenges in eukaryote functional genomics.

In future, a combination of RNAi and whole genome sequencing can contribute to the enhancement of the drug development success rates through better targets and RNAi platform efficiencies and keeping waste to a minimum by only treating people genetically predicted to respond to the therapeutic.

#### **Author details**

Gabriela N. Tenea and Liliana Burlibasa *Department of Genetics, University of Bucharest, Romania*

### **Acknowledgement**

GNT and LB were supported by The National Council of the Higher Education's Scientific Research (CNCSIS), IDEAS project: PNII 1958/2009.

#### **6. References**

manipulation.

**Author details**

**Acknowledgement**

**6. References**

338-342.

*Plant Cell* 2(4): 279–289.

**5. Conclusions**

The study of RNAi has led to a revolution in the understanding of gene expression and the examples in plants, animal and mammalian system as reviewed here, showed the diversity and the potential of RNAi as new approach to replaces the classical genetic technologies and

Today, the RNAi strategies as a new tool for cheap screen of gene function in organisms for which, a genetic approach was not developed yet. However, after 11 years of extensive research, RNAi has now been demonstrated to function in mammalian cells to alter gene expression and used as a means for genetic discovery as well as a possible strategy for

Finally, RNAi represent a significant tool for the accomplishment of these goals, and will undoubtedly be used to address many other challenges in eukaryote functional genomics.

In future, a combination of RNAi and whole genome sequencing can contribute to the enhancement of the drug development success rates through better targets and RNAi platform efficiencies and keeping waste to a minimum by only treating people genetically

GNT and LB were supported by The National Council of the Higher Education's Scientific

[1] Cogoni C, Macino G (2000) Post-transcriptional gene silencing across kingdoms. *Current*

[2] Mello CC, Darryl Conte Jr (2004) Revealing the world of RNA interference. *Nature* 431:

[3] Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans.

[4] De Haan P, Gielen JJL, Prins M, Wijkamp IG, Van Schepen A, Peters D, Van Grinsven MQJM, Goldbach R (1992) Characterization of RNA-mediated resistance to tomato

[5] Lindbo JA, Dougherty WG (1992) Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in

spotted wilt virus in transgenic plants. *Bio/Technology* 10: 1133-1137.

transgenic plants and protoplasts. *Virology* 189: 725-733.

genetic correction and genetic therapy in cancer and other diseases.

predicted to respond to the therapeutic.

Gabriela N. Tenea and Liliana Burlibasa

*Department of Genetics, University of Bucharest, Romania*

Research (CNCSIS), IDEAS project: PNII 1958/2009.

*Opinion in Genetic Development* 10(6): 638–643.

[27] Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in *Caenorhabditis elegans*. *Genome Biology* 2(1): 2.1–2.10.

RNAi Towards Functional Genomics Studies 89

[48] Rodriguez A, Griffiths-Jones S, Ashurst JL (2004) Identification of mammalian microRNA host genes and transcription units. *Genome Research* 14: 1902-1910. [49] Tang G, Galili G (2004) Using RNAi to improve plant nutritional value: from

[50] Ying SY, Lin SL (2004) Intron derived microRNAs-fine tunning of gene functions. *Gene*

[51] Zhao Y, Srivastava D (2007) A developmental view of microRNA function. *Trends*

[52] Lee RC, Feinbaum RL, Ambros V (1993) The *C. elegans* heterochronic gene lin-4 encodes

[53] Lagos-Quintana M, Rauhut R, Lendeckel W (2001) Identification of novel genes coding

[54] Lee RC, Ambros V (2001) An extensive class of small RNAs in *Caenorhabditis elegans.*

[55] Ketting RF, Fischer SEJ, Bernstein E, Sijen T, Hannon GJ, Plasterk RHA (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in

[56] Knight SW, Bass BL (2001) A role for the RNAse III enzyme DCR-1 in RNA interference

[57] Yekta S, Shih IH, Bartel DP (2004) MicroRNA-directed cleavage of HOXB8 mRNA*.*

[58] Yang M, Mattes J (2008) Discovery biology and therapeutic potential of RNA interference microRNA and antagomirs. *Pharmacology and Therapeutics* 117: 94-104. [59] Baskerville S, Bartel DP (2005) Microarray profiling of microRNAs reveals frequent

[60] Du TT, Zamore PD (2005) microPrimer: the biogenesis and function of microRNA.

[61] Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets including a stress-induced miRNA. *Molecular Cell* 14: 787-799. [62] Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-

[63] Park W, Li J, Song R, Messing J, Chen X (2002) CARPEL FACTORY: a Dicer homolog and HEN1 a novel protein act in microRNA metabolism in *Arabidopsis thaliana. Current*

[64] Peragine A, Yoshikawa M, Wu G, Albrecht HL, Poethig RS (2004) *SGS3* and *SGS2*/*SDE1*/*RDR6* are required for juvenile development and the production of *trans*-

[65] Vazquez F, Vaucheret H, Rajagopalan R, Lepers C, Gasciolli V, Mallory AC, Hilbert JL, Bartel DP, Crete P (2004) Endogenous trans-acting siRNAs regulate the accumulation of

[66] Aravin AA, Naumova NM, Tulin AV, Vagin VV, Rozovsky YM, Gvozdev VA (2001) Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable

mechanisms to application. *Trends In Biotechnology* 22(9): 463-469.

small RNAs with antisense complementary to lin-14*. Cell* 75: 843-854.

developmental timing in *C. elegans. Genes and Development* 15: 2654-2659.

and germ line development in *Caenorhabditis elegans. Science* 293: 2269-2271.

coexpression with neighboring miRNAs and host genes. *RNA* 11: 241-247.

associated small RNAs in plants*. Plant Cell* 14: 1605-1619.

*Arabidopsis* mRNAs. *Molecular Cell* 16: 69–79.

acting siRNAs in *Arabidopsis*. *Genes and Development* 18: 2368–2379.

elements in the *D. melanogaster* germline. *Current Biololy* 11: 1017–1027.

342: 25-28.

*Science* 294: 862-864.

*Science* 304: 594-596.

*Development* 132: 4645-4652.

*Biology* 12: 1484-1495.

*Biochemistry Science* 32(4): 189-97.

for small express RNAs. *Science* 294: 853-858.

[48] Rodriguez A, Griffiths-Jones S, Ashurst JL (2004) Identification of mammalian microRNA host genes and transcription units. *Genome Research* 14: 1902-1910.

88 Functional Genomics

[27] Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in

[28] Waterhouse PM, Helliwell CA, (2003) Exploring plant genomes by RNA induced gene

[29] Agrawal NP, Dasaradhi VN, Asif M, Pawan M, Bhatnagar RK, Mukherjee SK, (2003) RNA interference: biology mechanism and applications. *Microbiology and Molecular*

[31] Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in *Drosophila* cells. *Nature* 404 (6775): 293–296. [32] Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNAi

[33] Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. *Cell* 101(1): 25–33. [34] Voinnet O, Lederer C, Baulcombe DC (2000) Aviral movement protein prevents spread

[35] Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of

[36] Fjose A, Drivenes O (2006) RNAi and microRNAs: from animal models to disease

[37] Okamura K, Ishizuka A, Siomi H, Siomi MC (2004) Distinct roles for argonaute proteins in small RNA-directed cleavage pathways. *Genes and Development* 18: 1655-1666. [38] Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R (2005) Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. *Cell* 123: 631-640. [39] Kim DH, Behlke MA, Rose SD, Chang MS, Choi S, Ross J (2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. *Nature Biotechnology* 23: 222-226. [40] Siolas D, Lerner C, Burchard J, Ge W, Linsley PS, Paddison PJ, Hannon GJ, Cleary MA (2005) Synthetic shRNAs as potent RNAi triggers. *Nature Biotechnology* 23: 227-231. [41] Doench JS, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. *Genes*

[42] Miska EA (2005) How microRNAs control cell division differentiation and death.

[43] Alvarez-Garcia I, Miska EA, (2005) MicroRNA functions in animal development and

[44] Schiebel W, Pelissier T, Riedel L, Thalmeir S, Schiebel R, Kempe D, Lottspeich F, Sanger HL, Wassenegger M (1998) Isolation of an RNA-directed RNA polymerase-specific

[46] Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M (2005) Silencing of microRNAs in vivo with "antagomirs". *Nature* 438: 685-689. [47] Metzalapff M (2005) Applications of RNAi in crop improvement *Pflanzenschutz-*

*Caenorhabditis elegans*. *Genome Biology* 2(1): 2.1–2.10.

[30] Baulcombe D (2004) RNA silencing in plants. *Nature* 43: 356-363.

posttranscriptional gene silencing in plants. *Science* 286: 950-952.

of the gene silencing signal in *Nicotiana benthamiana Cell* 103: 157-167.

silencing. *Nature Reviews: Genetics* 4: 29-38.

plant microRNA targets*. Cell* 110: 513-520.

*Development* 17: 438-442.

*Nachrichten Bayer* 58(1): 51-59.

therapy. *Birth Defects Research (part C)* 78: 150-171.

*Current Opinion Genetic Development* 15: 563-568.

cDNA clone from tomato. *Plant Cell* 10: 2087-2101.

[45] Cullen BR (2005) RNAi the natural way. *Nature Genetics* 37:1163-1165.

human disease. *Development* 132: 4653-4662.

*Biology Reviews* 67: 4 657–685.

[67] Reinhart BJ, Bartel DP (2002) Small RNAs correspond to centromere heterochromatic repeats. *Science* 297: 1831.

RNAi Towards Functional Genomics Studies 91

[85] Jones L, Ratcliff F, Baulcombe DC, (2001) RNA-directed transcriptional gene silencing in plants can be inherited independently of the RNA trigger and requires Met1 for

[86] Matthew L (2004) RNAi for plant functional genomics. *Comparative And Functional*

[87] Hilson P, Allemeersch J, Altmann T, Aubourg S, Avon A, Beynon J, Bhalerao RP, Bitto F, Caboche M, Cannoot B, Chardakov V, Cognet-Holliger C, Colot V, Crowe M, Darimont C, Durinck S, Eickhoff H, Falcon De Longevialle A, Farmer EE, Grant M, Kuiper MTR, Lehrach H, Léon C, Leyva A, Lundeberg J, Lurin C, Moreau Y, Nietfeld W, Paz-Ares J, Reymond P, Rouzé P, Sandberg G, Dolores Segura M, Serizet C, Tabrett A, Taconnat L, Thareau V, Van Hummelen P, Vercruysse S, Vuylsteke M, Weingartner M, Weisbeek PJ, Wirta V, Wittink FRA, Zabeau M, Small I (2004) Versatile gene-specific sequence tags for *Arabidopsis* functional genomics: Transcript profiling and reverse

[89] Ifuku K, Yamamoto Y, Sato F (2003) Specific RNA interference in psbP genes encoded by multigene family in *Nicotiana tabacum* with a short 3'-untranslated sequence.

[90] Miki D, Itoh R, Shimamoto K (2005) RNA silencing of single and multiple members in a

[91] Yamamoto Y, Ifuku K, Sato F (2005) Suppression of psbP and psbQ genes in *Nicotiana tabacum* by RNA interference technique. In: van der Est A. Bruce D. (eds) *Photosynthesis: Fundamental Aspects to Global Perspectives*. Kluwers Academic Publisher The Netherlands

[92] Auer C, Frederick R (2009) Crop improvement using small RNAs: applications and

[93] Gheysen G, Vanholme B (2007) RNAi from plants to nematodes. *Trends Biotechnology* 25:

[94] Gordon KHJ, Waterhouse PM (2007) RNAi for insect-proof plants. *Nature Biotechnology*

[95] Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS (2006) Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic

[96] Wang M, Abbott D, Waterhouse PM (2000) A single copy of a virus derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. *Molecular Plant*

[97] Kusaba M, Miyahara K, Lida S, Fukuoka H, Takario T, Sassa H, Mischimura M, Nischio T (2003) Low-glutein content 1: a dominant mutation that suppresses the glutein

[98] Voelker T, Kinney AJ (2001) Variations in the biosynthesis of seed storage lipids. *Annual*

[99] Knowlton S (1999) Soybean oil having high oxidative stability. US Patent 5981781.

predictive ecological risk assessments. *Trends Biotechnology* 27: 644-651.

gossypol. *Proceedings of the National Academy of Sciences* 103: 18054-18059.

multigene family via RNA silencing in rice. *Plant Cell* 15: 1455-1467.

*Review Plant Physiolology. Plant Molecular Biology* 52: 335–361.

maintenance. *Current Biology* 11: 747–757.

genetics applications. *Genome Research* 14: 2176-2189. [88] Invitrogen Gatewaytm Technology. (2004) *Quest* 1(2): 32-33.

*Bioscience Biotechnology Biochemistry* 67: 107-113.

gene family of rice. *Plant Physiology* 138: 1903-1913.

*Genomics* 5: 240-244.

pp 798-799.

25: 1231-1232.

*Pathology* 1: 401-410.

89-92.

1833–1837.

123–132.

repeats. *Science* 297: 1831.

testes. *Nature* 442: 203–207.

plants. *Plos Biology* 2: E104.

development. *Genes and Development* 18: 1187-1197.

mediated by a transgene but not by a virus. *Cell 101*: 543-553.

RNA interference in *C. elegans*. *Current Biology* 10: 169-178.

pathway. *FEBS Letters* 579: 5822-5829.

4360-4364.

homologous to RNAdependent RNA polymerase. *Nature 399*: 166-169.

[83] Sato F (2005) RNAi and functional genomics. *Plant Biotechnology* 22: 431-442.

fungi. *Nature* 404: 245.

[67] Reinhart BJ, Bartel DP (2002) Small RNAs correspond to centromere heterochromatic

[68] Volpe TA, Kidner C, Hall IM, Teng G, Grewal SIS, Martienssed RA (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. *Science* 297:

[69] Mochizuki K, Gorovsky MA (2004) Small RNAs in genome rearrangement in

[70] Aravin A, Pfeffer GD, Lagos-Quintana M, Landgraf P, Iovino N, Morris P, Brownstein MJ, Kuramochi-Miyagawa S, Nakano T, Chien M, Russo JJ, Sheridan R, Sander C, Zavolan M, Tuschl T (2006) A novel class of small RNAs bind to MILI protein in mouse

[71] Catalanotto C, Azzalin G, Macino G, Cogoni C (2000) Gene silencing in worms and

[72] Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC (1999) The RDE-1 gene RNA interference and transposon silencing in *C. elegans. Cell* 99:

[73] Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in

[74] Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C (1998) AGO1 defines a novel locus of *Arabidopsis* controlling leaf development*. EMBO Journal* 17: 170-180. [75] Vaucheret H, Vasquez F, Crete P, Bartel DP (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant

[76] Zilberman D, Cao X, Jacobsen SE (2003) ARGONAUTE 4 control of locus-specific siRNA accumulation and DNA and histone methylation. *Science* 299: 716-719. [77] Kosik KS (2006) The neuronal microRNA system *Nature Reviews. Neurosciences* 7: 911-920. [78] Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC, (2000) An RNA-dependent RNA polymerase gene in *Arabidopsis* is required for posttranscriptional gene silencing

[79] Cogoni C, Macino G (1999) Gene silencing in *Neurospora crassa* requires a protein

[80] Smardon A, Spoerke JM, Stacey SC, Klein ME, Mackin N, Maine EM (2000) EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and

[81] Wu-Scharf D, Jeong B, Zhang C, Cerutti H (2000) Transgene and transposon silencing in *Chlamydomonas reinhardtii* by a DEAH-box RNA helicase. *Science 290*: 1159-1162. [82] Hammond SM (2005) Dicing and slicing: the core machinery of the RNA interference

[84] Tenea GN (2009) Exploring the world of RNA interference in plant functional genomics: a research tool for many biology phenomena. *Roumanian Biotechnological Letters* 14(3)

*Tetrahymena*. *Current Opinion in Genetic Development* 14: 181–187.

[100] Mroczka A, Roberts PD, Fillatti JJ, Wiggins BE, Ulmasov T, Voelker T (2010) An intron sense suppression construct targeting soybean FAD2-1 requires a double-stranded RNA-producing inverted repeat T-DNA insert. *Plant Physiology* 153: 882–891.

RNAi Towards Functional Genomics Studies 93

[117] Yu JY, Deruiter SL, Turner DL (2002) RNA interference by expression of short interfering RNAs and hairpin RNAs in mammalian cells *Proceedings of the National*

[118] Hannon GJ, Rossi JJ (2004) Unlocking the potential of the human genome with RNA

[119] Chi JT, Chang HY, Wang NN, Chang DS, Dunthy N, Brown PO (2003) Genome wide view of gene silencing by small interfering RNAs. *Proceedings of the National Academy of*

[120] McCaffrey AP, Nakai H, Pandey K, Huang Z, Salazar FH, Xu H, Wieland SF, Marion PL, Kay MA (2003) Inhibition of hepatitis B virus in mice by RNA interference. *Nature*

[121] Xia H, Mao Q, Eliason S, Harper SQ, Martins IH, Orr HT, Laulson HL, Yang L, Kotin RM, Davidson BL (2004) RNAi suppresses polyglutamine-induced neurodegeneration

[122] Palliser D, Chowdhury D, Wang Q, Lee SJ, Bronson RT, Knipe DM, Lieberman J (2006) An siRNA-based microbicide protects mice from lethal Herpex simplex virus 2 infection.

[123] Schiffelers RM, Xu J, Storm G, Woodle MC, Scaria PV (2005) Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis.

[124] Ryazansky SS, Gvozdev VA (2008) Small RNAs and cencerogenesis. *Biokhimia* 73(5):

[125] Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ (2005) RAS is regulated by the let-7 microRNA family.

[126] O'Connell RM, Taganov KD, Boldin MP, Cheng G, Baltimore D (2007) MicroRNA-155 is induced during the macrophage inflammatory response. *Proceedings of the National*

[127] Cheng AM, Byrom MW, Shelton J, Ford L (2005) Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis.

[128] Janot G, Simard MJ (2006) Tumour-related microRNAs functions in *Caenorhabditis*

[129] Kumar MS, Lu J, Mercer KL, Golub TR, Jacks T (2007) Impaired microRNA processing enhances cellular transformation and tumorigenesis. *Nature Genetics* 39: 673-677. [130] Calin GA, Ferracin M, Cimmino A, Di LG, Shimizu M, Wojcik SE, Iorio MV, Visone R, Sever NI, Fabbri M, Iuliano R, Palumbo T, Picchiorri F, Roldo C, Garzon R, Sevignani C, Rassenti L, Alder H, Volinia S, Liu CG, Kipps TJ, Negrini M, Croce CM (2005) A MicroRNA signature associated with prognosis and progression in chronic lymphocytic

[131] Saito Y, Liang G, Egger G, Friedman JM, Chuang JC, Coetzee GA, Jones PA (2006) Specific activation of microRNA-127 with downregulation of the proto-oncogene BCL6

by chromatin-modifying drugs in human cancer cells. *Cancer Cell* 9: 435-443.

leukemia. *The New England Journal of Medicine* 353: 1793-1801.

in a model of spinocerebellar ataxia. *Nature Medicine* 10: 816-820.

*Academy of Sciences* USA 99: 6047-6052.

interference. *Nature* 431: 371-378.

*Arthritis Rheumatology* 52: 1314-1318.

*Academy of Sciences* USA 104: 1604-1609.

*Nucleic Acids Research* 33: 1290-1297.

*elegans. Oncogene* 25: 6197-6201.

*Sciences* USA 100: 6343–6346.

*Biotechnology* 21: 639–644.

*Nature* 439: 89-94.

*Cell* 120: 635-647.

640-655.

[117] Yu JY, Deruiter SL, Turner DL (2002) RNA interference by expression of short interfering RNAs and hairpin RNAs in mammalian cells *Proceedings of the National Academy of Sciences* USA 99: 6047-6052.

92 Functional Genomics

*of Sciences* USA 108(1): 409-414.

virus X/potyviral synergism. *Virology* 231: 35-42.

replication of heterologous viruses*. Plant Cell* 9: 859-868.

*Genomics* 5: 240-244.

*Research* 102: 97-108.

*Plant Phatology* 5: 71-82.

*Development* 18: 1179-1186.

*Sciences* USA 95: 13079-13084.

RNA silencing. *Plant Cell* 16: 1235-1250.

transgene locus. *Plant Journal* 35: 82-92.

interference. *Nature* 418: 435–438.

*Biotechnology* 20: 500–505.

*Nature Medicine* 8: 681–686.

[100] Mroczka A, Roberts PD, Fillatti JJ, Wiggins BE, Ulmasov T, Voelker T (2010) An intron sense suppression construct targeting soybean FAD2-1 requires a double-stranded

[102] Matthew L (2004) RNAi for plant functional genomics. *Comparative And Functional*

[103] Shi XM, Miller H, Verchot J, Carrington JC, Vance VB (1997) Mutation in the region encoding the central domain of helper component-proteinase (HC-Pro) eliminate potato

[104] Pruss G, Ge X, Shi XM, Carrington JC, Bowman Vance V (1997) Plant viral synergism: the potyviral genome encodes broad–range pathogenicity enhancer that transactivates

[105] Roth BM, Pruss GJ, Vance VB (2004) Plant viral suppressors of RNA silencing. *Virus*

[106] Moissiard G, Voinnet O (2004) Viral suppression of RNA silencing in plants. *Molecular*

[107] Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja V, Carrington JC (2004) Viral RNA silencing suppressors inhibits the microRNA pathways at an intermediate step. *Genes*

[108] Dunoyer P, Lecellier CH, Parizotto EA, Himber C, Voinnet O (2004) Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of

[109] Anandalakshmi R, Pruss GJ, Ge X, Marathe R, Mallory AC, Smith TH, Vance VB (1998) A viral suppressor of gene silencing in plants. *Proceedings of the National Academy of*

[110] Kasschau KD, Carrington JC (1998) A counterdefensive strategy of plant viruses:

[111] Mallory AC, Mlotshwa S, Bowman LH, Vance VB (2003) The capacity of transgenic tobacco to send a systemic RNA silencing signal depends on the nature of the inducing

[113] Jacque JM, Triques K, Stevenson M (2002) Modulation of HIV-1 replication by RNA

[114] Lee NS, Dohjima T, Bauer G, Li H, Ehsani A, Salvaterra P, Rossi J (2002) Expression of small interfering RNAs targeted against HIV-1 *rev* transcripts in human cells.  *Molecular Therapy* 16: 378-386.

suppression of posttranscriptional gene silencing. *Cell* 95: 461-470.

[112] Downward J (2004) RNA interference. *British Medical Journal* 328: 1245-1248.

RNA-producing inverted repeat T-DNA insert. *Plant Physiology* 153: 882–891. [101] Hoffer P, Ivashuta S, Pontesc O, Vitins A, Pikaard C, Mroczka A, Wagnera N, Voelker T (2011) Posttranscriptional gene silencing in nuclei. *Proceedings of the National Academy*

[132] Esteller M (2007) Cancer epigenomics: DNA methylomes and histone-modification maps. *Nature Review Genet*ics 8: 286-298.

**Chapter 5**

© 2012 Bai, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License [http://creativecommons.org/licenses/by/3.0], which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Bai, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In their *Nature* paper, Fire and Mello wrote: 'Whatever their target, the mechanisms underlying RNA interference probably exist for a biological purpose'. Indeed, it's been shown that there are numerous cellular and physiological functions linked to RNAi [1]. For example, viral defense has been proposed to be the primary function of RNAi in both plants and flies [3]. In plants, virus infection could trigger sequence-specific gene silencing [4].

**Genome-Wide RNAi**

Hua Bai

http://dx.doi.org/10.5772/49945

**1. Introduction**

silencing (TGS) [2].

**Screen for the Discovery of**

Additional information is available at the end of the chapter

**Gene Function, Novel Therapeutical**

**Targets and Agricultural Applications**

The phenomenon of double-stranded RNAs (dsRNAs)-mediated gene silencing or RNA interference (RNAi] was first discovered in nematode *Caenorhabditis elegans* by Andrew Fire and Craig Mello in 1998 [1]. This great discovery gives rise to a fast-growing field and leads to the identification of novel RNAi pathways by which small interference RNAs (siRNAs) regulate gene expression and gene functions. Collective evidence suggests that the RNAi pathway is conserved in many eukaryotes and this pathway can be triggered by either exogenous or endogenous small interference RNAs. Exogenous dsRNAs (e.g. a virus with an RNA genome) are typically required a membrane transporter for dsRNA uptake into the cytoplasm, while endogenous small interference RNAs (e.g. microRNAs) are encoded in the genome. The precursors of both dsRNA and microRNA are first cleaved into short interference RNAs by a ribonuclease III (RNaseIII) enzyme, Dicer. Then these short interference RNAs initiate RNAi process when interacting argonaute proteins in the RNAinduced silencing complex (RISC). The small interference RNAs normally consist of 20~30 nucleotides. They can repress the transcription of message RNAs containing homologous sequences by either post-transcriptional gene silencing (PTGS) or transcriptional gene

## **Genome-Wide RNAi Screen for the Discovery of Gene Function, Novel Therapeutical Targets and Agricultural Applications**

Hua Bai

94 Functional Genomics

maps. *Nature Review Genet*ics 8: 286-298.

*Molecular Genetics* 13: R275-R288.

*Methods* 30: 313-321.

*Biology* 12: R852-R854.

USA 101: 6403-6408.

37: 911-924.

6: 443-453.

[132] Esteller M (2007) Cancer epigenomics: DNA methylomes and histone-modification

[133] Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, Jenuwein T, Livingston DM, Rajewsky K (2005) Dicer deficient mouse embryonic stem cell are defective in

[134] Buckingham SD, Esmaeili B, Wood M, Sattelle DB (2004) RNA interference: from model organisms towards therapy for neural and neuromuscular disorders. *Human*

[135] Kamath RS, Ahringer J (2003) Genome-wide RNAi screening in *Caenorhabditis elegans.*

[136] Eagle M, Boudouin SV, Chandler C, Giddings DR, Bullock R, Bushby K (2002) Survival in Duchenne muscular dystrophy: improvements in life expectancy since 1967 and the impact of home nocturnal ventilation. *Neuromuscular Disorders* 12: 926-929. [137] Goldman RD, Gruenbaum Y, Moir RD, Shumaker DK, Spann TP (2002) Nuclear lamins: building blocks of nuclear arhitecture. *Genes and Development* 16: 533-547. [138] Carthew RW (2002) RNA interference: the fragile X syndrome connection. *Current*

[139] Nollen EA, Garcia SM, van Haaften G, Kim S, Chavez A, Morimoto RI, Plasterk RH (2004) Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. *Proceedings of the National Academy of Sciences*

[140] Yang Y, Nishimura I, Imai Y, Takahashi R, Lu B (2003) Parkin suppresses dopaminergic neuron-selective neutotoxicuty induced by Pael-R in *Drosophila. Neuron*

[141] Caplen NJ, Taylor JP, Statham VS, Tanaka F, Fire A, Morgan RA (2002) Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA

[142] Kononenko NI, Shao LR, Dudek FE (2004) Riluzole-sensitive slowly inactivating sodium current in rat suprachiasmatic nucleus neurons. *Journal of Neurophysiology* 91: 710-718. [143] Smith RA, Miller TM, Yamanaka K, Monia BP, Condon TP, Hung G, Lobsiger CS, Ward CM, Wei H, Wancewicz Bennett CF, Cleveland DW (2006) Antisense oligonucleotide therapy for neurodegenerative disease*The Journal of Clinical Investigation* 116: 2290-2296. [144] Miller M, Kaspar KP, Kops GJ, Yamanaka K, Christian LJ, Gage FH, Cleveland DW (2006) Virus–delivered small RNA silencing sustain strength in amyotrophic lateral

[145] de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J (2007) Interfering with disease: a progress report on siRNA-based therapeutics. *Nature Reviews Drug Discovery*

[146] Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, Watts L, Booten S.L, Graham M (2006) miR-122 regulation of lipid metabolism revealed by in vivo antisense

interference. *Human Molecular Genetics* 11:175-184.

sclerosis. *Annual Neurology* 57: 773-776.

targeting. *Cell Metabolism* 3: 87-98

differentiation and centromeric silencing. *Genes and Development* 19: 489-501.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/49945

#### **1. Introduction**

The phenomenon of double-stranded RNAs (dsRNAs)-mediated gene silencing or RNA interference (RNAi] was first discovered in nematode *Caenorhabditis elegans* by Andrew Fire and Craig Mello in 1998 [1]. This great discovery gives rise to a fast-growing field and leads to the identification of novel RNAi pathways by which small interference RNAs (siRNAs) regulate gene expression and gene functions. Collective evidence suggests that the RNAi pathway is conserved in many eukaryotes and this pathway can be triggered by either exogenous or endogenous small interference RNAs. Exogenous dsRNAs (e.g. a virus with an RNA genome) are typically required a membrane transporter for dsRNA uptake into the cytoplasm, while endogenous small interference RNAs (e.g. microRNAs) are encoded in the genome. The precursors of both dsRNA and microRNA are first cleaved into short interference RNAs by a ribonuclease III (RNaseIII) enzyme, Dicer. Then these short interference RNAs initiate RNAi process when interacting argonaute proteins in the RNAinduced silencing complex (RISC). The small interference RNAs normally consist of 20~30 nucleotides. They can repress the transcription of message RNAs containing homologous sequences by either post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) [2].

In their *Nature* paper, Fire and Mello wrote: 'Whatever their target, the mechanisms underlying RNA interference probably exist for a biological purpose'. Indeed, it's been shown that there are numerous cellular and physiological functions linked to RNAi [1]. For example, viral defense has been proposed to be the primary function of RNAi in both plants and flies [3]. In plants, virus infection could trigger sequence-specific gene silencing [4].

© 2012 Bai, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License [http://creativecommons.org/licenses/by/3.0], which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Bai, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Plant RNAi forms the basis of virus induced gene silencing (VIGS), proofed from the genetic links between virulence and RNAi pathways [5-6]. On the other hand, endogenous microRNAs (about 1000 microRNAs in human genome) [7] play essential roles in controlling cellular functions. For example, the early discovered microRNAs, such as *lin-4* and *let-7* of *C. elegans*, were identified to regulate developmental timing [8-9]. Following the identification of *let-7* in *C. elegans* and later in fruit flies *Drosophila melanogaster* (hereafter I will refer it as '*Drosophila*'), it is soon realized that *let-7* belongs to a conserved microRNA family in many species. Besides the regulation on development, many microRNAs have been found to control key physiological processes, such as lipid metabolism [10] and insulin sensitivity [11]. The dysregulation of microRNA may result in many human diseases. A mutation in the seed region of miR-96 causes hereditary progressive hearing loss [12]. Some microRNAs have also been linked to cancer [13].

Genome-Wide RNAi Screen for the Discovery of

Gene Function, Novel Therapeutical Targets and Agricultural Applications 97

performed in *Drosophila* and mammalian cultured cells, as well as in primary cells. RNAi screen with *Drosophila* and mammalian cells has already led to important discovery in a wide variety of topics, including signal transduction, metabolism and cancer [22]. In general, a cell-based RNAi screen involves four major steps: [1]. RNAi library selection; [2]. Incubation of appropriate cell lines with RNAi reagents that are pooled or individually arrayed into 96- or 384-well plates; [3]. After additional treatments (if applicable), cells are subjected to the automated plate reader to quantify the specific readout (e.g. changes in cell morphology or fluorescence and luminescence signals from study targets); [4]. High-content

**RNAi library and reagent delivery methods.** Since the completion of *C. elegans* genome sequencing in 1998 [14], more and more eukaryotic genomes have been sequenced, which makes it possible to produce whole-genome RNAi libraries for functional genomics studies. Typically, long dsRNAs are used for RNAi screen in *Drosophila* cells, while synthetic siRNAs or vector-based short hairpin RNAs (shRNAs) are commonly used for mammalian cells [24]. Several *Drosophila* cell lines (e.g. S2 and Kc167) can directly take up dsRNA without the help of transfection reagents, which provides great advantages in high-throughput RNAi screens [25-26]. For mammalian cells, RNAi reagents are transient transfected into the cells. Therefore, not only the variation of transfection efficiency among cell lines and experimental replicates will affect the RNAi knockdown effects, but also the doubling time will affect the duration of gene silencing. Compared to synthetic siRNA method, vector-based shRNA technology combining with viral delivery methods provides robust gene silencing for a longer period of time. Besides, vector-based shRNA approaches make it possible to build

Now RNAi libraries are available for many model organisms, especially *Drosophila* and mammalian cells (Table. 1). These RNAi libraries normally contain the collection of RNAi reagents (dsRNAs, siRNAs and shRNAs) for all annotated genes in the genome. In the RNAi libraries, typically there are several different dsRNAs or siRNAs corresponding to each gene. For example, The GeneNet™ Human 50K siRNA library from System Biosciences contains 200,000 siRNA templates targeted to 47,400 human transcripts (~4 different siRNA sequences per transcript) (http://www.systembio.com/rnai-libraries). In *Drosophila* DRSC 2.0 RNAi collection, there are 1-2 dsRNAs per gene, including genes that encode proteins and non-coding RNAs (http://www.flyrnai.org). Beside genome-wide libraries, many pathway libraries or sub-libraries are also available for silencing specific signal pathways or multigene families (e.g. kinases & phosphatases library, G protein-coupled receptor library,

**Read-out assays.** Together with luminescent and fluorescent reporter-based image analysis and improved high-content image processing packages (e.g. CellProfiler [27] , various readouts are used in RNAi screens, including the expression changes in target genes or proteins, post-translation modification, metabolic processes, and changes in sub-cellular localization patterns. Most of these read-outs are based on the measurement of the intensity of luminescent and fluorescent reporters. Although RNAi screens for cell morphology (e.g. cytoskeletal organization and simple cell shape) have been previously reported [28-30],

image data analysis.

renewable and cost-effective RNAi libraries.

apoptosis & cell cycle library, etc.).

Soon after the discovery of dsRNA-mediated RNAi in 1998, gene silencing through RNAi was quickly developed as a powerful tool or technique in functional genomics studies. Comparing to forward genetics tools (e.g. EMS-induced mutagenesis screens), RNAi is one of effective reverse genetic tools, especially for non-model organisms and mammalian systems in which genetics is difficult. The advantage of applying RNAi in function studies becomes even more apparent when the whole genome sequences of model organisms (e.g. *C. elegans* and *Drosophila*) were completed in the late 90's and early 2000's [14-15]. In the post-genome era, utilizing high-throughput platforms and innovative bioinformatics tools, many large-scale RNAi screens has been successfully applied for the discovery of novel gene function associated with many important aspects of biology such as signal transduction [16], cell proliferation [17], metabolism [18], host-pathogen interactions [19] and aging [20-21]. Through these genome-wide RNAi screens, we have gained new insights on novel players in many key biological processes and complexity of cellular signaling networks. Cell-based and *in vivo* RNAi screen has been extensively reviewed in the past [22- 23].In this book chapter, I will focus on the recent development of high-throughput RNAi screen for functional analysis in cultured cells and *in vivo* systems, as well as its applications on functional genomics and the discovery of novel therapeutic drug and agricultural targets.

#### **2. RNAi screen methods**

Despite the challenges from off-target effects and false discovery during the data analysis, genome-wide RNAi screens have benefitted from improved RNAi delivery methods, automated high-content image system and robust statistic analysis [23]. In the following section, I will compare the different reagent delivery methods, read-out assays, and offtarget effects in various systems and platforms. I will also provide several examples from recent studies using genome-wide RNAi screen in cultured cells.

#### **2.1. Cell-based RNAi screen**

Genome-wide RNAi screen in cultured cells or primary cells provides an opportunity to systematically interrogate gene function. Now large-scale RNAi screens have been routinely performed in *Drosophila* and mammalian cultured cells, as well as in primary cells. RNAi screen with *Drosophila* and mammalian cells has already led to important discovery in a wide variety of topics, including signal transduction, metabolism and cancer [22]. In general, a cell-based RNAi screen involves four major steps: [1]. RNAi library selection; [2]. Incubation of appropriate cell lines with RNAi reagents that are pooled or individually arrayed into 96- or 384-well plates; [3]. After additional treatments (if applicable), cells are subjected to the automated plate reader to quantify the specific readout (e.g. changes in cell morphology or fluorescence and luminescence signals from study targets); [4]. High-content image data analysis.

96 Functional Genomics

microRNAs have also been linked to cancer [13].

**2. RNAi screen methods**

**2.1. Cell-based RNAi screen**

Plant RNAi forms the basis of virus induced gene silencing (VIGS), proofed from the genetic links between virulence and RNAi pathways [5-6]. On the other hand, endogenous microRNAs (about 1000 microRNAs in human genome) [7] play essential roles in controlling cellular functions. For example, the early discovered microRNAs, such as *lin-4* and *let-7* of *C. elegans*, were identified to regulate developmental timing [8-9]. Following the identification of *let-7* in *C. elegans* and later in fruit flies *Drosophila melanogaster* (hereafter I will refer it as '*Drosophila*'), it is soon realized that *let-7* belongs to a conserved microRNA family in many species. Besides the regulation on development, many microRNAs have been found to control key physiological processes, such as lipid metabolism [10] and insulin sensitivity [11]. The dysregulation of microRNA may result in many human diseases. A mutation in the seed region of miR-96 causes hereditary progressive hearing loss [12]. Some

Soon after the discovery of dsRNA-mediated RNAi in 1998, gene silencing through RNAi was quickly developed as a powerful tool or technique in functional genomics studies. Comparing to forward genetics tools (e.g. EMS-induced mutagenesis screens), RNAi is one of effective reverse genetic tools, especially for non-model organisms and mammalian systems in which genetics is difficult. The advantage of applying RNAi in function studies becomes even more apparent when the whole genome sequences of model organisms (e.g. *C. elegans* and *Drosophila*) were completed in the late 90's and early 2000's [14-15]. In the post-genome era, utilizing high-throughput platforms and innovative bioinformatics tools, many large-scale RNAi screens has been successfully applied for the discovery of novel gene function associated with many important aspects of biology such as signal transduction [16], cell proliferation [17], metabolism [18], host-pathogen interactions [19] and aging [20-21]. Through these genome-wide RNAi screens, we have gained new insights on novel players in many key biological processes and complexity of cellular signaling networks. Cell-based and *in vivo* RNAi screen has been extensively reviewed in the past [22- 23].In this book chapter, I will focus on the recent development of high-throughput RNAi screen for functional analysis in cultured cells and *in vivo* systems, as well as its applications on functional genomics and the discovery of novel therapeutic drug and agricultural targets.

Despite the challenges from off-target effects and false discovery during the data analysis, genome-wide RNAi screens have benefitted from improved RNAi delivery methods, automated high-content image system and robust statistic analysis [23]. In the following section, I will compare the different reagent delivery methods, read-out assays, and offtarget effects in various systems and platforms. I will also provide several examples from

Genome-wide RNAi screen in cultured cells or primary cells provides an opportunity to systematically interrogate gene function. Now large-scale RNAi screens have been routinely

recent studies using genome-wide RNAi screen in cultured cells.

**RNAi library and reagent delivery methods.** Since the completion of *C. elegans* genome sequencing in 1998 [14], more and more eukaryotic genomes have been sequenced, which makes it possible to produce whole-genome RNAi libraries for functional genomics studies. Typically, long dsRNAs are used for RNAi screen in *Drosophila* cells, while synthetic siRNAs or vector-based short hairpin RNAs (shRNAs) are commonly used for mammalian cells [24]. Several *Drosophila* cell lines (e.g. S2 and Kc167) can directly take up dsRNA without the help of transfection reagents, which provides great advantages in high-throughput RNAi screens [25-26]. For mammalian cells, RNAi reagents are transient transfected into the cells. Therefore, not only the variation of transfection efficiency among cell lines and experimental replicates will affect the RNAi knockdown effects, but also the doubling time will affect the duration of gene silencing. Compared to synthetic siRNA method, vector-based shRNA technology combining with viral delivery methods provides robust gene silencing for a longer period of time. Besides, vector-based shRNA approaches make it possible to build renewable and cost-effective RNAi libraries.

Now RNAi libraries are available for many model organisms, especially *Drosophila* and mammalian cells (Table. 1). These RNAi libraries normally contain the collection of RNAi reagents (dsRNAs, siRNAs and shRNAs) for all annotated genes in the genome. In the RNAi libraries, typically there are several different dsRNAs or siRNAs corresponding to each gene. For example, The GeneNet™ Human 50K siRNA library from System Biosciences contains 200,000 siRNA templates targeted to 47,400 human transcripts (~4 different siRNA sequences per transcript) (http://www.systembio.com/rnai-libraries). In *Drosophila* DRSC 2.0 RNAi collection, there are 1-2 dsRNAs per gene, including genes that encode proteins and non-coding RNAs (http://www.flyrnai.org). Beside genome-wide libraries, many pathway libraries or sub-libraries are also available for silencing specific signal pathways or multigene families (e.g. kinases & phosphatases library, G protein-coupled receptor library, apoptosis & cell cycle library, etc.).

**Read-out assays.** Together with luminescent and fluorescent reporter-based image analysis and improved high-content image processing packages (e.g. CellProfiler [27] , various readouts are used in RNAi screens, including the expression changes in target genes or proteins, post-translation modification, metabolic processes, and changes in sub-cellular localization patterns. Most of these read-outs are based on the measurement of the intensity of luminescent and fluorescent reporters. Although RNAi screens for cell morphology (e.g. cytoskeletal organization and simple cell shape) have been previously reported [28-30],

complex cell shape and structure-based read-outs still remain problematic. Frequently, additional treatments are performed before the read-out assays in RNAi screens. These treatments can be drugs, pathogens, or stress inducers [31-33].

Genome-Wide RNAi Screen for the Discovery of

Gene Function, Novel Therapeutical Targets and Agricultural Applications 99

signaling [37]; novel modulators of p53 pathway [38]; and key genes that are essential for the proliferation of cancer cells [17]. Recently, an integrative approach with RNAi screen and whole genome structural analysis identified IKBKE kinase as a breast cancer oncogene [39]. Beside its application in studying signaling pathways, cell-based RNAi screens are also performed to understand the cellular responses to pathogens. For example, recent RNAi screens identified novel host factors that are required for dengue virus propagation [40] and influenza virus replication [41]. I will discuss more detail on some of genome-wide RNAi

Many complex phenotypes, such as aging, cannot be tested in a cell-based assay, thus *in vivo* functional analysis is required*. In vivo* RNAi screen is one of such approaches to study gene function at an organism level. *C. elegans* and *Drosophila* are two model organisms that are commonly used in *in vivo* RNAi screens. Although *ex vivo* RNAi screens have been done by introducing shRNA-transfected cells into mice [42-43], direct *in vivo* RNAi screen in mice is still under development. Most importantly, *in vivo* RNAi makes it possible for gene function studies in species lacking classical genetic tools, even including species without whole genome sequences (Usually next-generation sequencing is used to identify gene coding

*In vivo* genome-wide RNAi screen was first reported in *C. elegans* [44]. In *C. elegans*, dsRNAmediated RNAi effects are systemic and heritable [1], although gene knockdown is less efficient in the nervous system than in other tissues. *In vivo* genome-wide RNAi screens have been performed in the studies of aging [20-21, 45-46], metabolism [47] and microRNA pathways in *C. elegans* [48]. It is relatively easy to deliver dsRNA into *C. elegans*. Typically, dsRNAs are introduced to worms by soaking the animals in dsRNA solution [49], by injection of dsRNA [1], or by feeding the animals dsRNA-expressing bacteria [44]. The last method is commonly used to generate genome-wide RNAi libraries. These *C. elegans* RNAi libraries are

In contrast, dsRNA feeding does not appear to work for gene silencing in *Drosophila*, while RNAi via injection of dsRNA is effective only in certain embryonic stages. Therefore, transgenic RNAi approach has been developed to express a double-stranded 'hairpin' RNA from a transgene. In *Drosophila*, RNAi is cell-autonomous, so that gene silencing can be easily performed in tissue- and spatial-specific manner by using the binary GAL4/UAS expression system. Currently, there are three groups that have generated genome-wide transgenic RNAi *Drosophila* stains [50] (Table. 2). These transgenic RNAi stains are all expressing invertedrepeat hairpin RNAs once crossing to appropriate Gal4 drivers. Recently, it's been shown that small hairpin RNAs (~19 nt) can trigger stronger gene inactivation than long hairpin RNAs. Therefore new constructs expressing small hairpin RNAs were generated to produce second generation of transgenic RNAi *Drosophila* stains at Harvard medical school (Table. 2). In the past several years, a number of genome-wide RNAi screens in *Drosophila* have been conducted to study the major signaling pathways [51], as well as many important disease models [52-55].

now available from Ahringer lab RNAi collection and Open Biosystems (Table. 2).

sequences and to facilitate RNAi reagent design and production).

screens in section. 3.

**2.2.** *In vivo* **RNAi screen**

**Table 1.** List of RNAi libraries used in cell cultures

**Off-target effects.** False positive or false negative results are commonly associated with high-throughput studies, including genome-wide RNAi screens [34]. The false discovery in RNAi screens can be caused by instrument errors, statistical noises, low knock-down efficiency and off-target effects of RNAi reagents. Off-target effects of RNAi reagents usually include: [1]. A general interference on endogenous RNAi pathway; or [2] Sequencedependent effects on the expression of non-target genes. In order to minimize off-target effects, it is suggested to perform sufficient replication experiments and choose two or more RNAi reagents that target different regions of the coding sequences. Sequence-dependent off-target effects can be avoided by selecting sequences that do not contain 19 or more base pairs of contiguous nucleotide identity to other genes in the genome [34-35].

**Recent cell-based RNAi screen studies.** Genome-wide RNAi screens have been primarily conducted in both *Drosophila* and mammalian cultured cells (Reviewed in [22]. These screens are involved in studies on a variety of biology processes, such as signal transduction, metabolism, cancer, stem cells. The cell-based RNAi screens have yielded tremendous amount of novel discoveries and greatly promoted our understanding on many basic biological processes, molecular functions and complexity of cellular networks. New findings from genome-wide RNAi screens have led to the identification of novel components of canonical signaling transduction pathways, such new players of ERK pathway [16] and phosphorylation networks regulating JUN NH2-terminal kinase (JNK) pathway [36]; the role of *S1pr2* gene (Sphingosine-1-phosphate receptor 2) in insulin signaling [37]; novel modulators of p53 pathway [38]; and key genes that are essential for the proliferation of cancer cells [17]. Recently, an integrative approach with RNAi screen and whole genome structural analysis identified IKBKE kinase as a breast cancer oncogene [39]. Beside its application in studying signaling pathways, cell-based RNAi screens are also performed to understand the cellular responses to pathogens. For example, recent RNAi screens identified novel host factors that are required for dengue virus propagation [40] and influenza virus replication [41]. I will discuss more detail on some of genome-wide RNAi screens in section. 3.

#### **2.2.** *In vivo* **RNAi screen**

98 Functional Genomics

*Genome-wide RNAi libraries*

*Pre-defined or custom RNAi libraries*

**Table 1.** List of RNAi libraries used in cell cultures

**Drosophila RNAi**

**The Netherlands**

complex cell shape and structure-based read-outs still remain problematic. Frequently, additional treatments are performed before the read-out assays in RNAi screens. These

**Name Species Type Link**

**Open Biosystems** Human, mouse siRNA , shRNA www.openbiosystems.com/R

**Sigma** Human, mouse siRNA , shRNA www.sigmaaldrich.com **SBI** Human, mouse siRNA , shRNA http://www.systembio.com/r

**Ambion** Human, mouse siRNA , shRNA www.invitrogen.com/sirna

**Cancer Institute (NKI)** Human shRNA www.lifesciences.sourcebiosc

**Off-target effects.** False positive or false negative results are commonly associated with high-throughput studies, including genome-wide RNAi screens [34]. The false discovery in RNAi screens can be caused by instrument errors, statistical noises, low knock-down efficiency and off-target effects of RNAi reagents. Off-target effects of RNAi reagents usually include: [1]. A general interference on endogenous RNAi pathway; or [2] Sequencedependent effects on the expression of non-target genes. In order to minimize off-target effects, it is suggested to perform sufficient replication experiments and choose two or more RNAi reagents that target different regions of the coding sequences. Sequence-dependent off-target effects can be avoided by selecting sequences that do not contain 19 or more base

**Recent cell-based RNAi screen studies.** Genome-wide RNAi screens have been primarily conducted in both *Drosophila* and mammalian cultured cells (Reviewed in [22]. These screens are involved in studies on a variety of biology processes, such as signal transduction, metabolism, cancer, stem cells. The cell-based RNAi screens have yielded tremendous amount of novel discoveries and greatly promoted our understanding on many basic biological processes, molecular functions and complexity of cellular networks. New findings from genome-wide RNAi screens have led to the identification of novel components of canonical signaling transduction pathways, such new players of ERK pathway [16] and phosphorylation networks regulating JUN NH2-terminal kinase (JNK) pathway [36]; the role of *S1pr2* gene (Sphingosine-1-phosphate receptor 2) in insulin

**Qiagen** Human, mouse siRNA , shRNA www.qiagen.com **Dharmacon** Human, mouse siRNA , shRNA www.dharmacon.com

pairs of contiguous nucleotide identity to other genes in the genome [34-35].

NAi

nai-libraries

ience.com/

**Screen Center** Fruit fly dsRNA www.flyrnai.org/ **DKFZ Genome RNAi** Fruit fly dsRNA www.genomernai.org

treatments can be drugs, pathogens, or stress inducers [31-33].

Many complex phenotypes, such as aging, cannot be tested in a cell-based assay, thus *in vivo* functional analysis is required*. In vivo* RNAi screen is one of such approaches to study gene function at an organism level. *C. elegans* and *Drosophila* are two model organisms that are commonly used in *in vivo* RNAi screens. Although *ex vivo* RNAi screens have been done by introducing shRNA-transfected cells into mice [42-43], direct *in vivo* RNAi screen in mice is still under development. Most importantly, *in vivo* RNAi makes it possible for gene function studies in species lacking classical genetic tools, even including species without whole genome sequences (Usually next-generation sequencing is used to identify gene coding sequences and to facilitate RNAi reagent design and production).

*In vivo* genome-wide RNAi screen was first reported in *C. elegans* [44]. In *C. elegans*, dsRNAmediated RNAi effects are systemic and heritable [1], although gene knockdown is less efficient in the nervous system than in other tissues. *In vivo* genome-wide RNAi screens have been performed in the studies of aging [20-21, 45-46], metabolism [47] and microRNA pathways in *C. elegans* [48]. It is relatively easy to deliver dsRNA into *C. elegans*. Typically, dsRNAs are introduced to worms by soaking the animals in dsRNA solution [49], by injection of dsRNA [1], or by feeding the animals dsRNA-expressing bacteria [44]. The last method is commonly used to generate genome-wide RNAi libraries. These *C. elegans* RNAi libraries are now available from Ahringer lab RNAi collection and Open Biosystems (Table. 2).

In contrast, dsRNA feeding does not appear to work for gene silencing in *Drosophila*, while RNAi via injection of dsRNA is effective only in certain embryonic stages. Therefore, transgenic RNAi approach has been developed to express a double-stranded 'hairpin' RNA from a transgene. In *Drosophila*, RNAi is cell-autonomous, so that gene silencing can be easily performed in tissue- and spatial-specific manner by using the binary GAL4/UAS expression system. Currently, there are three groups that have generated genome-wide transgenic RNAi *Drosophila* stains [50] (Table. 2). These transgenic RNAi stains are all expressing invertedrepeat hairpin RNAs once crossing to appropriate Gal4 drivers. Recently, it's been shown that small hairpin RNAs (~19 nt) can trigger stronger gene inactivation than long hairpin RNAs. Therefore new constructs expressing small hairpin RNAs were generated to produce second generation of transgenic RNAi *Drosophila* stains at Harvard medical school (Table. 2). In the past several years, a number of genome-wide RNAi screens in *Drosophila* have been conducted to study the major signaling pathways [51], as well as many important disease models [52-55].