RNAi screening and gene knockdown

Topics: RNA, DNA, Gene expression Pages: 11 (4101 words) Published: May 21, 2014
1. RNAi screening and gene knockdown
To put it in relatively simple terms, the experimental use of RNA interference (RNAi) permits researchers to determine the function of an individual gene, or a group of genes, or a protein encoded for by specific genes with a fairly straightforward procedure. This is done by silencing – or preventing – the expression of the gene in the developing or active cell during the transcription phase or the translation phase of gene expression. Research scientists use synthetically developed RNAi or plasmids in a procedure referred to as “gene knockdown.” Gene knockdown uses a physiological mechanism that inhibits the expression of genes by inducing the degradation of messenger RNA (mRNA). Messenger RNA is responsible for taking genetic commands and using them to code for production of amino acid chains and proteins by ribosomes. In the cells of mammals, RNAi is naturally mediated by Micro RNA (miRNA). miRNA are RNA transcripts that are produced by the cell similarly to normal messenger RNA, but that do not code for proteins or amino acids. Instead, miRNA are turned into shorter lengths of RNA. Unique to the physical structure of miRNA is a hairpin structure, called short hairpin RNA (shRNA). In turn, short hairpin RNA is processed into double stranded pieces of RNA referred to as short interfering RNA (siRNA). Inside of a complex of proteins called an RISC, or RNA Induced Silencing Complex, a single strand of an siRNA duplex will bind to a messenger RNA transcript that has a nucleotide sequence that is complementary to the sequence of the siRNA. This process permits an enzyme called nuclease in the RNA Induced Silencing Complex to slice and destroy the protein coding mRNA. By destroying the mRNA, the expression of the gene can be silenced in a manner that is specific to that sequence. The use of synthetically designed and produced short hairpin RNA and short interfering RNA has radically changed the manner in which researchers study the loss of function of specific genes or proteins. Before this technology was available, researchers were forced to use techniques that were far more time consuming, such as developing cell lines or animals with knockout sequences, referred to as “gene targeting.” Alternately, researchers could utilize techniques that were less time consuming but which produced unpredictable results, such as antisense RNA. Finally, researchers could specifically inhibit specific proteins, but these experiments were limited in their scope. Today, researchers can purchase RNAi reagents from molecular biology suppliers and contract research organizations (CROs), and use them to silence almost every gene in an organism’s genome. The degree to which the scope of gene knockdown research has been broadened will ultimately allow us to understand the function of virtually every gene in an organism’s genome, which has untold applications in clinical research and medicine. With the use of high throughput molecular applications, laboratories can test pools of genes at once, or even perform screens of an entire genome in a relatively short period of time. This breakthrough in gene knockdown research has revolutionized both the scale and speed with which gene and protein function is studied today.

2. RNAi applications in functional genomics
Functional genomics is a broad term for an area of molecular biology research that is geared towards developing an understanding of gene and protein functions and interactions. Where genomics uses molecular biology techniques to understand the sequence, function and structure of organisms’ entire genomes, functional genomics is interested in the active processes of translation and transcription of individual genes. The majority of applications using RNA interference in animals have used the model organisms C. elegans and Drosophilia melanogaster. These organisms are commonly used because RNAi has been shown to have the most effective results when...
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