Go to the following link to gain information about RNA Interference technology h

Question

Go to the following link to gain information about RNA Interference technology
http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html

Click on the link for “RNAi Explained”

9.  What is thought to be the natural “purpose” of the RNAi system when it first evolved in primitive organisms?

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Briefly describe the roles of Dicer and RISC in the RNAi process. Include which acts first.

Ultimately, the translation of a specific mRNA is blocked in the RNAi process. How is this blockage step achieved?

Explanation / Answer

Genetic loss of function studies in murine tumor models have been essential in the analysis of downstream mediators of oncogenic transformation. Unfortunately, these studies are frequently limited by the availability of genetically modified mouse strains. Here we describe a versatile method allowing the efficient expression of an oncogene and simultaneous knockdown of targets of interest (TOI) from a single retroviral vector. Both oncogene and TOI specific miR30-based shRNA are under the control of the strong viral LTR promoter, resulting in a single shared RNA transcript. Using this vector in a murine syngeneic bone marrow transplantation model for BCR-ABL induced chronic myeloid leukemia we find that oncogene expression and target knockdown in primary hematopoietic cells with this vector is efficient both in vitro and in vivo, and demonstrate that Raf1, but not BRAF, modulates BCR-ABL dependent ERK activation and transformation of hematopoietic cells. This expression system could facilitate genetic loss-of-function studies and allow the rapid validation of potential drug targets in a broad range of oncogene driven murine tumor models. The original picture is this: in response to dsRNA, cells trigger a two-step reaction. In the first step, long dsRNA is processed by a ribonuclease (RNase) III enzyme called Dicer into small interfering RNAs (siRNAs); these subsequently serve as the sequence determinants of the RNAi pathway by directing cleavage of homologous mRNA via an RNA-induced silencing complex (RISC) (Reviewed in Hannon [2002]). Similar small RNA molecules that can silence gene activity are micro-RNAs (miRNAs). These single-stranded RNA molecules, which have widespread roles in growth and development, are also the result of Dicer activity, but in this case, the stem loop precursor molecules are encoded within the animal or plant genomes, and silencing can occur either via destruction of the mRNA (plants) or by blocking its translation (animals and plants) (see Carrington and Ambros [2003] for a recent review). Much insight into the mechanism of RNAi has come from biochemical analysis within Drosophila cells and extracts. Forward genetic screens performed in other genetic systems, such as C. elegans, Neurospora, and Arabidopsis have led to the identification of crucial players in the RNAi pathway; unfortunately, however, the biochemistry for these systems does not, at present, match the level of finesse reached with Drosophila in vitro systems. Carthew and colleagues (Lee et al., 2004 [this issue of Cell]) now bring the two approaches together; these authors used genetically engineered Drosophila strains to identify mutants that had either a reduced or an enhanced response to eye-specific expression of a hairpin dsRNA corresponding to white sequences (white null mutants have white eyes whereas wild-type Drosophila eyes are red) and then analyzed them biochemically. There is a good correlation between the number of transgenes that express the hairpin and the level of white silencing (arguing against trigger amplification in this animal): one copy turns the eye pale orange, but two copies result in white eyes; this allows a combined enhancer and suppressor mutant search by screening animals that contain a single copy. Importantly, the screen is based on a mosaic-generating system that produces homozygous mutant compound eyes in an otherwise heterozygous animal. The ability to score RNAi phenotypes of mutations in essential genes (which when homozygous would result in lethality or sterility) seems crucial for further identification of RNAi components, considering the mechanistic overlap in the RNA silencing pathways triggered by siRNAs and miRNAs and the crucial regulatory roles of miRNAs in growth and development. This separation of function for Drosophila Dicers is, however, not absolute; although it remains to be seen whether Dcr-2 has any contribution in miRNA function (what do the double mutant look like?), Dcr-1 is certainly also required for efficient RNAi. Surprisingly, the role of Dcr-2 in RNAi is not limited to processing long dsRNA into siRNAs. By direct injection of synthetic siRNAs into Drosophila eggs, one can bypass the dsRNA-processing activity of Dicers. Whereas wild-type eggs exhibited a profound RNAi response, dcr-2 null mutant eggs displayed an impaired response, implying a role for Dcr-2 downstream of siRNA formation, backing up recent observations made in mammalian cells where siRNAs failed to induce RNAi upon cotransfection of siRNAs directed against Dicer (Doi et al., 2003). Interestingly, the helicase domain present in Dcr-2 (but not in Dcr-1) that is required for dicing activity is dispensible for siRNA-induced mRNA degradation. Our ongoing research into optimizing RNA analysis has identified two points in the RNA isolation process that can be improved; treatment and handling of tissue or cells prior to RNA isolation and storage of the isolated RNA. Since most of the actual RNA isolation procedure takes place in a strong denaturant (e.g. GITC, LiCl, SDS, phenol) that renders RNases inactive, it is typically prior to, and after the isolation, when RNA integrity is at risk. Finding the most appropriate method of cell or tissue disruption for your specific starting material is important for maximizing the yield and quality of your RNA preparation. See the article "Cell Disruption - Getting the RNA Out," which describes various disruption methods and suggests which method to use for specific tissues / cell types, for more information on this subject. During tissue disruption for RNA isolation, it is crucial that the denaturant be in contact with the cellular contents at the moment that the cells are disrupted. This can be problematic when tissues/cells are hard (e.g. bone, roots), when they contain capsules or walls (e.g. yeast, Gram-positive bacteria) or, when samples are numerous, making rapid processing difficult. A common solution to these problems is to freeze the tissue/cells in liquid nitrogen or on dry ice. The samples must then be ground with a mortar and pestle into a fine powder, which is added to the denaturant. While this freezing and grinding process allows the researcher to postpone RNA isolation, it is a time consuming and laborious process. Use of RNAlater for tissue storage is compatible with most RNA isolation procedures. Tissues stored in RNAlater are simply removed and processed by homogenization via a dounce, Polytron® (Brinkman), or other mechanical apparatus in the lysis buffer specified by your RNA isolation procedure. See the article "Cell Disruption - Getting the RNA Out" for a discussion of sample disruption. Figure 2 shows the RNA isolated from tissue stored in RNAlater Solution using several methods, and Figure 3 demonstrates that mRNA signal intensities in RPAs are not affected by storage in RNAlater.

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