**3. Mechanism of gene silencing**

The purpose of gene silencing is to regulate gene expression by degrading the targeted gene's product or reducing its mRNA translation through the delivery of RNAi agents into the cytoplasm [28]. Gene silencing mechanism involves both exogenous and endogenous pathways. The silencing mechanism of synthetic RNAi agents such as siRNA, shRNA, and miRNA is through the exogenous pathway, whereas the silencing mechanism for miRNA that exists naturally in the cell is through the endogenous pathway [29]. The mechanism of these three synthetic RNAi agents depends on the RNA-induced silencing complex (RISC) to cleave or degrade the mRNA of the targeted gene.

Further process of siRNA silencing occurs in the cytoplasm, whereas for shRNA and pre-miRNA, DNA integration occurs in the nucleus prior to the changes of pri-miRNA/pri-shRNA to the pre-miRNA/pre-shRNA [30]. The siRNA pathway is activated when dsRNA, together with the carrier complex, penetrates the cell membrane. Then, an endogenous dicer enzyme identifies the dsRNA sequence and splits it into small fragments of siRNA. The RISC complex binds to the siRNA fragments and causes RNA splitting for gene silencing to occur [31]. Plasmid carried by the lentiviral vector encodes the shRNA sequence of the

targeted gene and also shRNA transcripts on the promoter of RNA pol III or pol II. When the lentivirus infects the cells, lentiviral plasmids are transferred to the cytoplasm. An endogenous dicer enzyme identifies the plasmid and splits the shRNA into small fragments of pri-shRNA [32]. The small fragments of prishRNA enter the nucleus, are multiplied by reverse transcription, and integrate into the host cell's genome. The integrated genome will enter the shRNA silencing pathway to silence the gene.

Pre-shRNA and microRNA primers (pri-miRNA) are transcribed in the nucleus using a complex of Drosha microprocessors and DGCR8 as intermediators to produce the precursor microRNA (pre-miRNA)/pre-shRNA. Pre-miRNA/preshRNA is then exported to the cytoplasm by exportin-5 and splits by a dicer enzyme to dsRNA [33]. The dsRNA is combined with RISC and is resolved by a helicase. The disassembled dsRNA activates the mRNA thread guide to recognize the target gene and Argonaute protein (Ago) in RISC [34]. The RISC complex helps to locate the mRNA thread guide which is complementary to the mRNA molecules of the targeted gene. The targeted gene's mRNA is then degraded by the endonuclease and inactivated [35].

#### **3.1 Critical factors of gene silencing**

Each cell has a different level of susceptibility toward lentivirus infection, and this is the biggest challenge to overcome in order to achieve optimal conditions for successful transduction [36]. The efficiency of shRNA silencing on the targeted gene is measured manually by experiment. Therefore, several critical factors must be considered to achieve specificity and efficiency of cell transduction. The factors are cell density, polybrene concentration, serum presence in the transduction media, incubation time, and lentivirus and puromycin dosage [37]. Furthermore, the functional titer and the multiplicity of infection (MOI) should be determined so that the minimum amount of lentivirus needed to transduce the cell is used. The ideal shRNA structure and experimental design should also be taken into account [38].

#### **3.2 Basic conditions of gene silencing**

One of the basic conditions that needs to be optimized for successful gene silencing is the cell density. The recommended cell density for transduction is 40–50% of cell confluence [16]. However, it depends on the size and growth rate of the cell. This confluence is to ensure that the cells have enough space to divide during transduction, which takes about 96 h. A previous study showed that cell confluence of more than 50% limits the interaction of lentivirus, cells, and DNA complex [39]. On the other hand, cell confluence of less than 30% will slow down cell growth [40]. Besides that, the cells used for transduction process must be active. This is because active cells take up foreign molecules more efficiently compared to quiescent cells.

The lentivirus membrane and cell wall are negatively charged. This causes difficulty for the lentivirus to infect the cell. Polybrene is a cation polymer that facilitates the lentivirus's infiltration into the cells [41]. Higher concentration of polybrene results in a more effective lentiviral infection. However, if the concentration of polybrene is too high and it is incubated with the cells for a long duration, it may cause toxic effects to the cells [42]. Therefore, a polybrene concentration that does not affect cell viability in long culture periods was chosen as the optimum condition. Studies have reported that the absence of serum in the culture media increases the efficiency of lentivirus DNA uptake by the cell [43]. However,

**107**

any false positive.

*Silencing of Peroxiredoxin-4 in Anticancer Activity of Gamma-Tocotrienol*

the absence of serum may affect cell growth. Therefore, the presence of serum in the media during the transduction process has to be determined for efficient

cells will die as puromycin inhibits protein synthesis [44]. Cells are cultured in media containing different concentrations of puromycin for 7 days. Then, a kill curve is constructed. The optimal dosage of puromycin to be used is the lowest dosage of puromycin that kills the cell significantly from the third to the seventh day. Cell viability is calculated starting from the third day because the cells only respond

the functional titer needs to be determined in order to know the minimum lentivirus concentration required to infect the cell. Functional titer is the smallest transducing unit required for lentivirus to infect the cell. The transducing unit needs to be parallel to the ratio between the lentivirus and cell in culture, which is known as MOI. Functional titer and MOI are important to ensure optimum condition for shRNA expression for transduction to occur. Technically, only a small concentration of functional titer is required to transduce cells at a consistent MOI ratio [45]. MOI is the ratio of lentivirus that can infect the cell at one time. As the MOI increases, the lentivirus volume required in a fixed amount of functional titer also increases. The minimum MOI should be determined so that the optimum concentration of shRNA that is sufficient to infect the cells at the recommended

Stable silenced cells are selected through puromycin resistance. Non-transduced

Each cell type has different susceptibilities to lentivirus infection. Therefore,

The design of shRNA vector is important to control the optimum expression of shRNA. The optimum expression of shRNA is determined by the lentiviral vector, promoters, and shRNA structures [47]. A study reported that human cytomegalovirus (hCMV) promoter is able to infect 95% of HepG2 cells and stably integrate into the host cell genome [48]. Lentivirus infection has low toxicity and does not affect the phenotype of the cell [49]. An efficient combination of promoter and vector will have an influence on the functional titer. Thus, shRNA can be expressed optimally

A good experimental design is essential in gene silencing so that the silencing effect can be validated as specific silencing without any false positive. The recommended experimental design for transduction method comprises a positive control, a negative control, and unsilenced cell [50]. The positive control is a sequence of normalized genes that are highly expressed in cells to identify whether the silencing have effects on the cell cycle or any pathways that may affect the growth of the silenced cells [51]. The presence of nontargeted lentivirus negative control (NTC) is to validate whether the plasmid of the lentivirus itself has an effect on the targeted gene expression. NTC is consists of shRNA sequence that is not complementary to any mammalian gene by having three or less nucleotide equivalents. It also contains GFP and also puromycin-resistance sequences. This control is important to avoid

The NTC control should have no effect on the targeted gene and the transduced cell [52]. The non-transduced cell controls are standardized to be compared with NTC controls. If the NTC and non-transduced cell controls show no difference in mRNA and protein expression of targeted proteins, this proves that

*DOI: http://dx.doi.org/10.5772/intechopen.88813*

to the antibiotic exposure after 48 h.

confluence is used [46].

**3.3 Small hairpin RNA (shRNA) design**

to specifically silence a target gene.

**3.4 Experimental design**

transduction.

*Silencing of Peroxiredoxin-4 in Anticancer Activity of Gamma-Tocotrienol DOI: http://dx.doi.org/10.5772/intechopen.88813*

*Synthetic Biology - New Interdisciplinary Science*

pathway to silence the gene.

**3.1 Critical factors of gene silencing**

**3.2 Basic conditions of gene silencing**

compared to quiescent cells.

inactivated [35].

account [38].

targeted gene and also shRNA transcripts on the promoter of RNA pol III or pol II. When the lentivirus infects the cells, lentiviral plasmids are transferred to the cytoplasm. An endogenous dicer enzyme identifies the plasmid and splits the shRNA into small fragments of pri-shRNA [32]. The small fragments of prishRNA enter the nucleus, are multiplied by reverse transcription, and integrate into the host cell's genome. The integrated genome will enter the shRNA silencing

Pre-shRNA and microRNA primers (pri-miRNA) are transcribed in the nucleus

Each cell has a different level of susceptibility toward lentivirus infection, and this is the biggest challenge to overcome in order to achieve optimal conditions for successful transduction [36]. The efficiency of shRNA silencing on the targeted gene is measured manually by experiment. Therefore, several critical factors must be considered to achieve specificity and efficiency of cell transduction. The factors are cell density, polybrene concentration, serum presence in the transduction media, incubation time, and lentivirus and puromycin dosage [37]. Furthermore, the functional titer and the multiplicity of infection (MOI) should be determined so that the minimum amount of lentivirus needed to transduce the cell is used. The ideal shRNA structure and experimental design should also be taken into

One of the basic conditions that needs to be optimized for successful gene silencing is the cell density. The recommended cell density for transduction is 40–50% of cell confluence [16]. However, it depends on the size and growth rate of the cell. This confluence is to ensure that the cells have enough space to divide during transduction, which takes about 96 h. A previous study showed that cell confluence of more than 50% limits the interaction of lentivirus, cells, and DNA complex [39]. On the other hand, cell confluence of less than 30% will slow down cell growth [40]. Besides that, the cells used for transduction process must be active. This is because active cells take up foreign molecules more efficiently

The lentivirus membrane and cell wall are negatively charged. This causes difficulty for the lentivirus to infect the cell. Polybrene is a cation polymer that facilitates the lentivirus's infiltration into the cells [41]. Higher concentration of polybrene results in a more effective lentiviral infection. However, if the concentration of polybrene is too high and it is incubated with the cells for a long duration, it may cause toxic effects to the cells [42]. Therefore, a polybrene concentration that does not affect cell viability in long culture periods was chosen as the optimum condition. Studies have reported that the absence of serum in the culture media increases the efficiency of lentivirus DNA uptake by the cell [43]. However,

using a complex of Drosha microprocessors and DGCR8 as intermediators to produce the precursor microRNA (pre-miRNA)/pre-shRNA. Pre-miRNA/preshRNA is then exported to the cytoplasm by exportin-5 and splits by a dicer enzyme to dsRNA [33]. The dsRNA is combined with RISC and is resolved by a helicase. The disassembled dsRNA activates the mRNA thread guide to recognize the target gene and Argonaute protein (Ago) in RISC [34]. The RISC complex helps to locate the mRNA thread guide which is complementary to the mRNA molecules of the targeted gene. The targeted gene's mRNA is then degraded by the endonuclease and

**106**

the absence of serum may affect cell growth. Therefore, the presence of serum in the media during the transduction process has to be determined for efficient transduction.

Stable silenced cells are selected through puromycin resistance. Non-transduced cells will die as puromycin inhibits protein synthesis [44]. Cells are cultured in media containing different concentrations of puromycin for 7 days. Then, a kill curve is constructed. The optimal dosage of puromycin to be used is the lowest dosage of puromycin that kills the cell significantly from the third to the seventh day. Cell viability is calculated starting from the third day because the cells only respond to the antibiotic exposure after 48 h.

Each cell type has different susceptibilities to lentivirus infection. Therefore, the functional titer needs to be determined in order to know the minimum lentivirus concentration required to infect the cell. Functional titer is the smallest transducing unit required for lentivirus to infect the cell. The transducing unit needs to be parallel to the ratio between the lentivirus and cell in culture, which is known as MOI. Functional titer and MOI are important to ensure optimum condition for shRNA expression for transduction to occur. Technically, only a small concentration of functional titer is required to transduce cells at a consistent MOI ratio [45]. MOI is the ratio of lentivirus that can infect the cell at one time. As the MOI increases, the lentivirus volume required in a fixed amount of functional titer also increases. The minimum MOI should be determined so that the optimum concentration of shRNA that is sufficient to infect the cells at the recommended confluence is used [46].

### **3.3 Small hairpin RNA (shRNA) design**

The design of shRNA vector is important to control the optimum expression of shRNA. The optimum expression of shRNA is determined by the lentiviral vector, promoters, and shRNA structures [47]. A study reported that human cytomegalovirus (hCMV) promoter is able to infect 95% of HepG2 cells and stably integrate into the host cell genome [48]. Lentivirus infection has low toxicity and does not affect the phenotype of the cell [49]. An efficient combination of promoter and vector will have an influence on the functional titer. Thus, shRNA can be expressed optimally to specifically silence a target gene.

#### **3.4 Experimental design**

A good experimental design is essential in gene silencing so that the silencing effect can be validated as specific silencing without any false positive. The recommended experimental design for transduction method comprises a positive control, a negative control, and unsilenced cell [50]. The positive control is a sequence of normalized genes that are highly expressed in cells to identify whether the silencing have effects on the cell cycle or any pathways that may affect the growth of the silenced cells [51]. The presence of nontargeted lentivirus negative control (NTC) is to validate whether the plasmid of the lentivirus itself has an effect on the targeted gene expression. NTC is consists of shRNA sequence that is not complementary to any mammalian gene by having three or less nucleotide equivalents. It also contains GFP and also puromycin-resistance sequences. This control is important to avoid any false positive.

The NTC control should have no effect on the targeted gene and the transduced cell [52]. The non-transduced cell controls are standardized to be compared with NTC controls. If the NTC and non-transduced cell controls show no difference in mRNA and protein expression of targeted proteins, this proves that

the plasmid carried by the lentivirus has no effect on the cell. Although shRNA functions are at the mRNA level, only changes in protein expression may contribute to phenotypic changes. Therefore, validation of the mRNA and protein expression of the targeted proteins should be carried out to ensure that the gene expression is suppressed [53]. In addition, it is essential to validate the silencing effect by using more than two sequences of targeted gene shRNA. Every segment of gene sequence has a different effect in silencing the gene. The best sequence is selected based on the higher silencing rates shown through reduction of mRNA and protein expression of targeted gene.
