RNAi+Lab+Prospectus

// The Function of RNAi // // Background of RNAi //  RNAi (RNA interference), discovered by Andrew Fire and Craig Mello in 1998, is a cellular mechanism naturally occurring in eukaryotic organisms. The occurrence of RNAi has many tremendous applications, such as treating cancer or other diseases that produce malevolent proteins. Through RNAi, specific genes are turned off, or silenced. RNAi is predicated upon the involvement of dsRNA, which breaks down the mRNA for a specific gene (thus 'silencing it,' or turning it off). Because the process of RNAi silences a particular gene’s expression, it stops the production of a specific protein.

In order to initiate RNAi, both sense + antisense RNA must be present. Sense + antisense bond to form dsRNA, which is the initial step in the RNAi process, as previously explained. Basically, sense RNA is a copy of a particular mRNA segment (that codes for some protein). Antisense RNA is a complement to a specific mRNA; it binds to the specific mRNA, thus inactivating it. For more information, please refer to [|(>^.^)>]

When the dsRNA (double-stranded RNA) binds with dicer protein (an enzyme occurring within cells), it is cut up into numerous segments (siRNA, small interfering RNA). The RISC ( RNA-induced silencing complex) complex is then formed when RISC binds to siRNA, and the siRNA is thus unzipped. These products are ‘active RISC’ and bind to a target mRNA- thus prohibiting the mRNA from being transcribed into a functional protein. Functional proteins are synthesized based upon exact codes- which unaltered mRNA contain. However, when the 'active RISC' bind to a target mRNA, they interfere with this 'code' being read- thus an incorrect protein is formed, which cannot function as its physical composition has been altered.

In cells, precursors of MicroRNA form hairpin structures of dsRNA; these molecules activate RNAi "machinery." After RNAi occurs, the particular gene is silenced as its genetic information (DNA) doesn’t come to fruition- the transcription of a protein is blocked. Thus dsRNA regulates gene expression since it blocks a particular protein (coded for by the corresponding gene) from being created.

Simplification of the before-mentioned process: RNAi is a term describing a particular way of silencing a gene. If a gene is silenced, then proteins associated with it will not be made correctly. For instance, if we put dsRNA specific to a gene that synthesizes a needed protein in cancer cells, the cancer cells would stop growing and die. Isolating the code associated with said protein is difficult though...



// Our Experiment with C. elegans and the Bli-1 Gene //

The goal of our class experiment was to silence the Bli-1 gene, which codes for 'normal cuticle functioning' in //C. elegans//. If the Bli-1 gene in //C. elegans// is silenced through RNAi, then the affected worms will express visible blisters. Dr. Bacay, of wormbase.org, mentions, "The bli-1 gene encodes an unusual cuticular collagen that is required for proper strut formation within the unique medial layer of the adult cuticle; bli-1 interacts genetically with other cuticular collagens such as bli-2 and rol-1, and may be processed for secretion by BLI-4, a Kex2/subtilisin serine endoproteinase; consistent with its role in adult cuticle formation, bli-1 mRNA is highly expressed only during the L4 larval stage."

Our experiment entailed many processes which are all explained on our 'RNAi Experiment Components' page, found here. In order to initiate the mechanism of RNAi in //C. elegans//, we needed to create the dsRNA corresponding to the mRNA of the Bli-1 gene and get it inside of the //C. elegans//. Since //C. elegans// is a relatively uncomplicated organism (nematode), we would be able to transform cells (HT115) of the growth medium for //C. elegans// with a vector containing an insert of a portion of the Bli-1 gene.

The portion of the Bli-gene, when replicated in //C. elegans//, will form dsRNA, thus initiating RNAi. Yet, in order to obtain the Bli-1 gene, we needed to lyse open //C. elegans// and extract its genomic DNA. By utilizing PCR, we were able to amplify the segment of Bli-1 numerous times. We bought the vector L4440, isolated it, and ligated it with our amplified Bli-1 in order for the HT115 cells to be transformed by it. This simplified procedure that was just outlined required many different procedures and confirmations, which can be found on our 'RNAi Experiment Components' page:

-Worm Care and Feeding - Dr. Andrew P. Radocchia -Isolation of Genomic DNA - Dr. Caroline L. Nightingale, PhD. -PCR Amplification of Gene of Interest - Dr. Christopher M. Lee -The Creation of the Cloning Vector Using L4440 - Dr. Alyssa F. Bacay, PhD. -Transformation of DH5a then HT115 - Dr. Alanna F. DeRogatis, PhD. -Feeding Transformed HT115 to Worms - Dr. Jeremy M. Rosenblatt

In order to view our experimental results, please refer to each individual page (linked above).

// Sources //

Source of Bli-1 information found: @http://www.wormbase.org/db/gene/gene?name=WBGene00000251;class=Gene

Additional RNAi resources found here.