Discovery of the EVI-1 gene


The aim of the project is to investigate potential EVI-1 target genes. These genes were primarily discovered by microarray analysis in Rat1 cells ectopically expressing the EVI-1 gene. The transforming activity of EVI-1 may changes in gene expression (thus potentially be involved in the development of cancer) they could be secondary changes involved in the transformation process (not directly target genes of EVI-1) or they might be genes that are regulated by EVI-1. The identification of target genes of the EVI-1 was observed by microarray analysis in the Rat1 transformation assay. In this study will examine one potential gene that is differentially expressed based upon the microarray data and will confirm its differential expression using alternative techniques to microarrays. Also investigate the activity of the promoter of this potential target gene if it is regulated by EVI-1 and investigate the biological processes that EVI-1 is normally involved in by using inhibitory RNAs. Rat 1 fibroblasts are transformed by overxpreesion of EVI-1, which is likely to be linked to its role in leukaemia.

Discovery of the EVI-1 gene:

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Evi-1 (ectopic viral integration site 1) was first identified as a common site of retroviral integration in myeloid tumours in AKXD mice (Mucenski et al, 1988). Mice from 21 of 23 AKXD recombinant innate mouse strains derived from crossing AKR/J, a strain with a high occurrence of lymphoma, with DBA/2J, a strain with a low occurrence of lymphoma, develop haematopoietic neoplasmas at a high incidence. DNA of AKXD lymphomas that include single somatically acquired proviruses were characterised to conclude whether new common sites of integration could be identified that present new proto-oncogene Ioci involved in haemopoietic diseases. Evi-1 was consequently discovered and encodes a 145KDa nuclear localised transcription factor with two zinc finger domains. Also discovered by it is activation in IL-3 dependent murine myeloid cell line isolated from retrovirous induced leukemias (Morishita et al, 1988). In humans, the EVI-1gen ortholog located on chromosome 3, band q24-q28 (Morishita et al, 1990). The gene spans over 100 kb and contains 21 exons with 10 coding exons (Suzukawa et al., 1997). Mouse and human EVI-1sequences show 91%and 94% nucleotide and amino acid homology, respectively (Morishita et al., 1990).

Structure of the EVI-1 protein:

The structure of ecotropic viral integration site-1 (EVI-1) and myelodysplastic syndrome (MDS) 1-EVI-1 show in figure 1. The EVI-1 gene encodes 1051 amino acids DNA binding phosphoprotein, it is a C2 H2 zinc finger family proteins with transcriptional repressor activity that is dependent on a repressor domain RP (Bartholomew et al., 1997). The 145 kDa EVI-1 protein is localized in the nucleus (Matsugi et al., 1990), and has two sets of the zinc finger domains (ZF) and between the two sets of zinc finger domains, a repression domain (RD) has been discovered including an acidic region at the C-terminus (Goyama and Mineo., 2009).

The EVI-1 gene is transcribed into mRNA with several variant5'-end (Aytekin et al, 2005). In both human and mouse, EVI-1 has an alternative spliced forms. One spliced variants, designated, Δ324 that lacks the zinc fingers 6and 7 including the transcription repression domain. (Kilbey and Bartholomew, 1998). Another Evi-1 mRNA variant, Δ105 is truncated by 105 amino acids at its C-terminus and has reduced activity in the Rat-1 fibroblast transformation assay. This mRNA variant was abundant in murine cells, but was not detected in human cell. In addition to alternative splicing that use different transcriptional initiation sites and contains a 188 amino acid extension, which is designated MDS1 or PR domain, at the 5 end of the formerly reported EVI-1 protein. MDS1/ EVI-1 (ME) created from the in-frame splicing of the small gene myelodysplasia syndrome 1 (MDS1) to the second exon of EVI-1 (Fears et al., 1996). MDS1 itself was first identified because it is reorganized in a 3; 21 translocation and was mapped approximately 300 kb upstream of EVI-1. This MDS1 domain is a proline-rich region of the gene (Wieser, 2007). The PR domain has homology with SET domain, which associated with histon methyltransferrase activity. SET domains are found at the c-terminus of the protein, while PR domains are located at the n-terminus (Nucifora et al, 2006). Relatively, the PR domain in MDS1-EVI-1 prevents oligomerization, which affects its biochemical functions (Nitta et al, 2005).

Biochemical properties of the EVI-1 protein:

EVI-1 protein shows that it functions as a transcriptional factor of the zinc finger family. It consists of N-terminal seven- zinc finger domain (ZF1) and C-terminal three -zinc finger domain (ZF2). The multiple fingers are necessary to obtain high-affinity, site-specific DNA binding. ZF1 binds to a consensus sequence GA(C/T) AAGA (T/C) AAGATAA and ZF2 binds to the consensus sequence GAAGATGAG (Bartholomew et al, 1997; Jolkowska and Michal, 2000; Nucifora et al, 2006). The carboxyl domain contains three continuous zinc fingers while in the amino terminal the first zinc finger is separated from the others by 25 amino acids. Only four (4-7) zinc fingers of the first domain are necessary for DNA binding, the remaining ones do not bind DNA but assist in binding the DNA. Binding through finger 9 is important for the DNA binding ability of the carboxyl domain. Mutations of the zinc finger sequences prevent and completely eliminate binding.

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EVI-1 interacts with transcriptional co-activator and co-repressor (Chakraborty et al, 2001; Izutsu et al, 2001; Vinatzer et al, 2001). The co-repressors that interact with EVI-1 include C-terminal binding protein (CtBP) and the histone deacetylases (HDACs). CtBP was originally identified as a protein that interacts with the C-terminal region of the adenoviral oncoprotein E1A through the 5-amino acid sequences PLDLS and PVDLS Bind (Schaeper, 1995). EVI-1 consists of one CtBP binding consensus motif (PLDLS) in the distal end. PLDLS site of EVI-1 physically interacts with CtBP and CtBP represses the transcription of a reporter gene by EVI-1(Chakraborty et al., 2001; Izutsu et al., 2001). However, EVI-1 has also been shown to interact with HDAC1 and HDAC4 directly and through sites different from those required for CtBP binding (Plamer et al., 2001; Vinatzer et al., 2001). EVI-1 is obviously a complex transcription factor with various functions, and this complexity is further confirmed by the ability of EVI-1 to interact with co-activators, particularly P/CAF at the N-terminus, and CBP/ p300 in the central region. The main biochemical function of EVI-1 is that of a transcription factor (Buonamici et al., 2003).

Biological function of EVI-1:

EVI-1 and CtBP interactions:

CtBP was shown to be important in biological activity, as point mutations in this site reduced the ability of EVI1 to transform Rat-1 fibroblasts (Palmer et al., 2001) and to inhibit reporter gene activation as well as growth inhibition of cells in response to TGF-β.

The interaction between this oncoprotein and CtBP is biologically important and appears to be involved in growth deregulation and abnormal differentiation.

This indicates that point mutations in CtBP eliminated certain biological activities of EVI-1 in steadily transfected cells. (Palmer et al., 2001

The mechanistic basis of the prosurvival and proproliferative activities of CtBPs

We discovered that CtBPs are required for the maintenance of mitotic fidelity. In addition to being a previously unidentified cellular role for these proteins, this is an important component of their prosurvival function.

Evi-1 need repressor domain to transform Rat 1 fibroblast:

Evi-1 encodes a transcriptional repressor and has significant inferences for the method of action of the Evi-1 protein both in development and in the progression of some myeloid leukaemias. The Rat1 transformation assay was applied to study the essential of the repressor domain to the activity of the Evi-1 protein while, recombinant retroviral vectors have evi-1 full length cDNA or a deletion mutant cDNA, were created. The repressor domain identified by deletion mutagenesis is important to the biological activity of the Evi-1 protein (Bartholomew et al., 1997).

EVI-1 and cell proliferation.

Several lines of evidence indicate that Evi-1 is involved in cell proliferation. Evi-1 may promote the cell cycle and inhibit cell death, by the fact that many body regions of the Evi-1 knocout mice were hypocellular (Hoyt et al., 1997). Evi-1 accelerated the cell cycle of Rat-1 fibroblasts (Bartholomew et al., 1997; Kilbey et al., 1999), of the murine myeloid cell line 32Dcl3 (Chakraborty et al., 2001; Chi et al., 2003), and of murine embryonic stem (ES) cells. In Rat-1 fibroblasts, Evi-1 overexpression was associated with lowered p27 protein levels, increased cdk2 activity, and increased levels of hyperphosphorylated Rb protein, as well as a shortened G1 phase of the cell cycle (Kilbey et al., 1999). Gata-2 was discovering as an essential Evi-1 target gene in the cell cycle (Yuasa et al., 2005). Several authors reported that EVI-1 accelerated the cell cycle of myeloid progenitors from murine bone marrow, whereas proliferation of erythroid cells was inhibited by EVI-1 (Wieser, 2007).

Chi et al. (2003) demonstrated that Evi-1 also interacts with BRG1 to upregulate cell cycling. BRG1 is a member of the SWI/SNF chromatin-remodeling complex and is a positive regulator of RB in cell cycle. This relatively recent report shows that Evi-1 activates the E2F1 promoter in 3T3 cells and upregulates cell cycling in BRG1-positive cells, but not in BRG1-negative cells. Evi-1 promotes cell proliferation in the BRG1-positive cells.

EVI-1 effect on haemopoietic differentiation:

Evi-1 has been revealed to have effect on haemopoietic cells where its expression blocks terminal differentiation. Over expression of Evi-1 in the 32Dc13, an interleukin-3 dependent myeloid cell line that differentiates in response to granulocyte colony-stimulating factor (G-CSF) does not alter the normal growth factor requirement of the cells. However, the cells were unable to terminally differentiate in response to G-CSF. The overexpression of Evi-1 in myeloid cells should interfere with the cells ability to terminally differeniate (Morishita et al., 1992). The Gata-1 transcription factor is essential for differentiation along the erythroid lineage. EVI-1 interacted directly with Gata-1 through the N-terminal zinc finger domain of the earlier protein and the C-terminal zinc finger of the later protein (Laricchia-Robbio et al., 2006). EVI-1 reduced the activity of Gata-1 dependent erythroid differentiation including myeloid differentiation of 32Dc13 cells (Vinatzeret al., 2001). The forced expression of EVI-1 has been studied in undifferentiated and differentiated murine ES cells. EVI-1 increases growth rate and cause differentiation along the megakaryocytic lineage (Sitailo et al., 1999).

EVI-1 effect on programmed cell death:

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Several evidences have been illustrated, EVI-1 as a survival factor that counteracted a diversity of apoptotic stimuli. Antisense repression of EVI-1 improved ultraviolet (UV) light provoked cell death of HEC1B endometrial adenocarcinoma cells. However, over expression of EVI-1 blocked UV induced apoptosis of human fetal kidney and Jurkat acute T-cell leukemia cells including tumor necrosis factor (TNF)-α induced apoptosis of histiocytic lymphoma cells (Kurokawa et al., 2000). Also EVI-1 protected murine bone marrow progenitor cells and SiHa cervival carcinoma cells from apoptosis in response to interferon (IFN)-α, an agent that is used in the therapy of CML (Buonamici et al., 2005).

EVI-1 and signalling pathways:

EVI-1 represses TGF-β signalling through recruitment of CtBP.

EVI-1 has been shown to affect different signalling pathways. TGF-β is the best-characterized pathway that is impeded by EVI-1. TGF-β plays an essential role in tumor development and controls cell proliferation. EVI-1 inhibits TGF-β signalling repressing its growth inhibition in cells. EVI-1 interacts with Smad3 and the corepressor CtBP to inhibit TGF-β signalling. In contrast, MDS1-EVI-1 increase TGF-β induced growth inhibition in 32D cells and cannot suppress TGF-β activity (Goyama and Kurokawa, 2009; Buonamici et al, 2003).

EVI-1 inhibits JNK-induced apoptosis.

EVI-1 blocks cell death by selectively inhibiting c-Jun N-terminal kinase (JNK), thus contributing to the oncogenic transformation of cells. JNK is implicated in the stress response of cells and it leads to apoptosis. EVI-1 represses JNK by interfering in the interaction between JNK and its substrates (Kurokawa et al, 2000). EVI-1 physically interacts with JNK through the ZF1 domain. By inhibiting JNK activity, EVI-1 protects cells from stress-induced cell death, therefore contributing to malignant transformation (Buonamici et al, 2003; Nucifora et al, 2006). In addition, EVI-1 suppresses TGF-β or taxol-mediated apoptosis through a phosphoinositid 3-kinase (PI3K)-Akt dependent mechanism in RIE cells (Liu et al., 2006).

EVI-1 abrogates effects of INFα.

EVI-1 can repress the effect of the cytokines such as, INFα is a cytokine that controls the immune response and limitations expansion of different tissue as well as bone marrow. Generally, EVI-1 specifically represses IFNα-dependent initiation of the tumour suppressor PML, thus blocking the apoptotic path way of PML. This discovers new mechanism used by oncogene to escap the normal cell response to growth- controlling cytokines (Buonamici et al., 2005; Nucifora et al., 2006).

Normal EVI-1 gene expression:

Several investigators have considered the pattern of normal EVI-1 expression during murine embryonic development and in adults. EVI-1 is expressed at high levels in several embryonic mouse tissues include the urinary system and Mullerian ducts, the bronchial epithelium of the lung, the focal area within the nasal cavities, the endocardial cushions and truncus swellings in the heart and the developing limbs. However, EVI1 is expressed at low level in most adult mouse tissues due to the spatial and temporally restricted pattern of expression of EVI1, it was suggested that this gene plays an essential role in organogenesis and morphogenesis in mouse development (Buonamici et al., 2004; Nucifora et al., 2006; Wieser., 2007). The mutant of embryos mice exhibited multiple malformations, which included widespread hypocellularity, a reduced body size, a pale yolk sac and placenta, defects in the heart and peripheral nervous systems fails to develop. This suggests that EVI-1 has an essential role in general cell proliferation, vascularization and cell-specific developmental at midgestation (Wieser, 2007). In the hematopoietic cells, EVI1 is expressed at low level early in myeloid cell differentiation, but is radically increased at promyelocytic stage (Buonamici et al., 2003). There are sources showing the EVI-1 expression in normal bone marrow ad also can be expressed in highly purified CD34+ progenitor cells (Privitera et al., 1997).

EVI-1 and Leukaemia.

Leukemias are the most frequent malignant diseases in children. Most cancers, including leukemias are caused by genomic alterations which accumulate within cellular DNA and modify their activity. The most frequent chromosomal abnormalities as inversions and translocations. Some chromosomal translocations can create novel chimeric genes (Jolkowska and Michal, 2000). Inappropriate expression of EVI-1 in hematopoietic cells has been associated with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The activation of EVI-1 often occurs through chromosome 3q26, translocations and inversions that stimulate the constitutive expression of the gene and presumably lead to development or progression of the leukemia (Zoccola et al., 2003). The chromosomal breakpoints at 3q26 in the translocation are mapped 5' of EVI-1, whereas the breakpoint in the inversion cases are located 3' of EVI-1. In both cases, the deregulation of EVI-1 is forced by the enhancer elements associated with the RibophorinI gene (Buonamici et al., 2003; Nucifora et al., 2006). EVI-1 is also activated in some ovarian cancers suggesting a possible role in solid tumour development (Brook et al., 1996). EVI-1 can be expressed in early hematopoietic progenitor cells. This suggests that abnormal EVI-1 expression may contribute to the leukemic phenotype by interfering with other genes which control the commitment of progenitors to proliferation and differentiation (Jolkowska and Michal, 2000). Expression of EVI-1 in patients with chronic myelocytic leukaemia in blast crisis (CML-BC) is higher than in other types of leukemia. These facts show that EVI-1 expression may be used to indicate transformation from chronic phase to blast crisis in patients (Jolkowska and Michal, 2000). Patients who inappropriately express EVI-1 often have monosomy of chromosome 7 or deletion of the long arm of chromosome 7, less frequently, deletions in chromosome 5. The most characteristic clinical features of these patients are elevated platelet counts, marked hyperplasia with dysplasia of megakaryocytes, and poor prognosis (Buonamici et al., 2003).

Occurrence of fusion mRNA and fusion transcription as a result of chromosomal translocation comprises of one critical element of leukaemogensis (Mitani et al., 1994).

The AML1-MDS1-EVI1 (AME) is the chimeric gene product of the (3; 21) (q26; q22) translocation linked with de novo and therapy-related MDS AML and CML-BC. The AML1 gene, also known as RUNX1, encodes the DNA-binding alpha subunit of the core-binding transcription factor (CBF) (Senyuk et al,2002). Recently, it was shown that AME induces myeloid leukemia in mice either alone (Cuenco et al, 2000) or in cooperation with BCR-ABL (Cuenco et al., 2004). It is most important to examine the role of EVI-1 in the pathogenesis of human leukaemias. Many studies of EVI-1 function in malignant blood cells including normal cells in detection of target genes of the EVI-1 protein are critical for understanding the role of this gene (Jolkowska and Michal, 2000).

Target genes of the EVI-1:

A number of potential target genes of Evi-1 were identified through microarray analysis and chromatin immunoprecipitation studies. These putative target genes discovered include Gadd45g, Gata2, Zfpm2/Fog2, Ski1 (SnoN), K1f5 (BTEB2), Dcn and Map3k14 (Nik) (Yatsula et al., 2005).

Activator protein (AP)-1 is a factor transcription complex that comprises of a Fos-Jun heterodimer or Jun-Jun homodimer. AP-1 is controls a number of cellular processes as well as differentiation, proliferation, and apoptosis. In NIH-3T3 and P19 cells, EVI-1 stimulates AP-1 activity and triggers endogenous c-Jun and c-Fos with dependence on its distal zinc finger domain. As the distal zinc finger domain is essential for EVI-1-mediated transformation of Rat-1 cells, the improved of AP-1 activity perhaps lead to cell transformation by EVI-1 (Tanaka et al., 1994; Goyama and Kurokawa, 2009).

Carbonic anhydrase III (caIII) is an essential antioxidant in normal liver and it may be critical in protecting haemopoietic cell from oxidative stress. EVI-1 shown to be represses of the expression of caIII this repression is directly or indirectly alters transcription of caIII gene in Rat1 cells. This lead to improved sensitivity to hydrogen peroxide (H2O2) induced apoptosis in Rat1 cells (Roy et al., 2009).

Gata-2 is a critical target gene of EVI-1 that plays a role in both maintenance and proliferation of HSCs. EVI-1 is important in the transcriptional regulation of Gata-2 in HSCs. EVI-1 directly binds to GATA-2 promoter as an enhancer. The activation of Gata-2 contributes to EVI-1 induced block to myeloid and erythroid differentiation and cell proliferation (Yuasa et al., 2005; Yatsula et al., 2005).

PbX1 is a proto-oncogene in hematopoietic malignancy and a target gene of Evi-1. Pbx1 expression is elevated in hematopoietic stem cells by over expression of Evi-1 Pbx1. An analysis of promoter region of Pbx1 illustrated that Evi-1 up regulates Pbx1 transcription. In addition, decrease of Pbx1 levels through RNAi-mediated knockdown repressed Evi-1-induced transformation. However, knockdown of Pbx1 did not empire bone marrow transformation via E2A/HLF or AML1/ETO, suggesting that Pbx1 is particularly essential for the maintenance of bone marrow transformation mediated by Evi-1 (Shimabe et al., 2009).

MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in various cellular processes as well as hematopoiesis and in pathogenesis of AML. Several authors have been found there is link between miRNAs and AML cryptogenic subgroup. EVI-1 expression correlated with the expression of miR-1-2 and miR-133-a-1 in both cell lines and in patient samples. EVI-1 attaches directly to the promoter of these two miRNAs. However, only miR-1-2 was complicated in abnormal proliferation but not miR-133-a-1. In addition, miR-133-a-1 may be play role in inhibiting of differentiation. (Gomez-Bento et al., 2010).


Luciferase report assays:

Genetic reporters are used as indicators to study gene expression and cellular events coupled to gene expression. In general, a reporter gene is cloned with a DNA sequence of interest into an expression vector that is then transferred into cells. After that, the cells are examined for the presence of the reporter using direct reporter measurement of the protein itself or the enzymatic activity of the reporter protein. A good reporter gene can be identified easily and measured quantitatively when it is expressed. Bioluminescence includes a number of diverse chemistries developed for light production and is based on the interaction of the enzyme, luciferase, with a luminescent substrate, luciferin (Allard et al., 2008).

Preparation of total RNA and protein:

The NucleoSpin RNA/Protein method, a solution containing large amounts of chaotropic ions in incubation used to lysis cells. This lysis buffer immediately inactivates virtually all enzymes which are present in almost all biological materials. The buffer dissolves proteins allowing them to pass NucleoSpin column, produces appropriate conditions for binding RNA to the silica membrane. Contamination DNA that binds to the silica is removed by an rDNase solution. Simple washing with two different buffers remove salts, metabolites and macromolecularcellular components. Pure RNA is finally eluted under low ionic strength conditions with RNase-free water. Protein is isolated from the column flow-through. Protein is precipitate in denatured form with a special buffer which effectively precipitates protein. After a washing step the protein pellet is dissolved in protein solving Buffer containing the odourless reducing agent TCEP.

Preparation of Plasmid DNA:

The bacteria are resuspended and SDS/alkaline lysis used to liberate plasmid DNA from E.coli host cells (Buffer A2). The resulting lysate is neutralized by Buffer A3 and forms suitable situation for binding of plasmid DNA to the silica membrane of the Nucleospin Plasmid or Nucleospin Plasmid Quickpure Column. In centrifugation step, the precipitated protein, genomic DNA, and cell debris are pelleted. Then the NucleoSpin Plasmid or NucleoSpin Plasmid QuickPure Column used to load the supernatant. After that, simple washing with ethanoic used to remove contamination like salts, metabolites and soluble macromolecular cellular components from the NucleoSpin Plasmid kit. Finally, pure plasmid DNA eluted under low ionic strength conditions with slightly alkaline Buffer AE. Additional washing with preheated Buffer AW is used if host strains have high levels of nucleases. This washing will elevate the reading length of automated fluorescent DNA sequencing reactions.

Preparation of cDNA:

SuperScript™ III Reverse Transcriptase is used to synthesis cDNA at a temperature range of 42-60°C, offering improved specificity, higher yield of cDNA, and more full-length product than other reverse transcriptases. SuperScript™ III Reverse Transcriptase involved in the RT Enzyme Mix to decrease RNase H activity and offered enlarged thermal stability. Ribosomal and transfer RNA do not repress SuperScript™ III RT, result in synthesize cDNA from total RNA. RNaseOUT™ Recombinant Ribonuclease Inhibitor also added in the enzyme mix against the degradation of target RNA owing to ribonuclease contamination of the RNA preparation. The 2X RT Reaction Mix contain oligo(dT)20, random hexamers, MgCl2, and dNTPs in a buffer formulation that has been optimized for qRT-PCR. Then, RNA template eliminate from the cDNA:RNA fusion molecule after first-strand synthesis by E. coli RNase H that is supplied as a separate tube in the kit.

QPCR assay:

ABsolute TM Fast QPCR Mix and ROX Vial have been used for DNA and c DNA quantification. This 2X mix controls all the mechanism that a chive a fast, sensitive and reproducible QPCR reaction, with the probability of the primers and template. Thermo- FastTM DNA polymerase has been added to avoid non- specific amplification during the reaction. Proprietary reaction buffer offers highly sensitive, specific and regular fluorescence readings for real-time and end-point analysis. This buffer comprises of MgCl2 and enhancers to progress amplifications of wide range template as well as DNA, GC rich fragments and inert blue dye has been added to support visualization. Additionally, dNTPs and dTTP have enhanced reaction sensitivity and efficiency compared to dUTP. ROX is also added for data normalization.

DNA Transfection:

FuGENE 6 Transfection Reagent is moderate on the cells. The DNA:FuGENE 6 reagent complex used to transfect adherent cells before plating, creating it a strong candidate for elevated throughput applications. FuGENE 6 Transfection Reagent can simply be utilized to transfect low cell numbers in 96-well plates. The complex formation is created by diluting the FuGENE 6 Transfection Reagent in serum free medium, and then DNA is added to the diluted. In the cells, the complex is added directly without change. Then the cells are returned to the incubator until the time of the gene-expression assay (Jacobsen et al., 2003).


In cells, enzymatic cleavage of long dsRNAs used to produce small interfering RNAs (siRNAs) via RNase-III class endoribonuclease Dicer. The siRNAs connect with the RNA Induced Silencing Complex (RISC) in a method that is assisted by Dicer. Dicer-Substrate RNAi processes are benefit in the link between Dicer and RISC loading that happens when RNAs are formulated by Dicer. The TriFECTa kit comprise of three Dicer-Substrate 27-mer duplexes targeting a particular gene that are chosen from a predesigned set of duplexes from the RefSeq group of human, mouse, and rat genes in Genbank. In addition to three target-specific duplexes, the TriFECTa kit have three control sequences that are required to achieve RNAi experiments involved a Cy3 TM -labeled transfection control (Cy3 TM DS Transfection Control) RNA duplex, a negative control RNA duplex that is missing in human, mouse, and rat genomes, and a positive control Dicer-Substrate RNA duplex.


Call cells and might suggest the basis for the development of a novel therapeutic strategy for the treatment of leukaemias and solid tumours where EVI-1 is overexpressed (Roy et al., 2009).