Even prior the establishment of RNAi in operating in mammalian systems, its potential as a potent research tool was already recognized. With the onset of genomic sequencing experiments of humans and other common model organisms resulted to a large amount of genetic identity with little knowledge on their function [18]. An economical approach in ablating gene function holds out a great prospect for the improvement of human ability to unlock the complexity of the regulatory pathways that control cellular behavior.
RNAi pathway enables scientists to conduct analysis on the effect of loss of gene function at the cellular level in a short period of time, which would otherwise cause several months had other methods been employed [2]. RNAi is a significant method in targeting individual genes. It is currently a frequent choice in biological and biomedical researches to study the effect of blocking expression of a given gene. In studies on mammals, the in vitro introduction of small interfering double stranded RNAs into cells of tissue culture is the main concentration [17].
However, mammalian cells will not spontaneously take up naked nucleic acids, they would have to be complexed with agents in order to allow their entry into the cell. siRNAs artificially synthesized can cause efficient inhibition of expression of homologous genes but only for a limited number of days. This results to incomplete effects, giving it the term “knock down” to distinguish it from the “knock out” that is achieved in the deletion of the gene [5].
Other methods of siRNA introduction into cellular systems is the use of vectors such as engineered viruses to direct short RNA sequences that will encode for hairpins owing to the presence of complementary sequences of about 20 nucleotide pairs. The mentioned shRNAs are then processed inside the cellular environment to remove the loop and form siRNA duplexes. This viral vector mediated RNA interference can cause gene expression inhibition for a long period of time. Exaples of viruses used as vectors are retroviruses, adenoviruses, and lentiviruses [5]. Figure 3. RNA Interference Targeting Disease [10]
There are different limitations on RNA interference despite the improvements done on this procedure. First is that not all the sequences function properly, researchers only reach success rates of about 33%. Even if effects are assumed to be sequence specific, it still cannot be determined well whether some or not some of the effects seen are “off-target. ” Some residual activation of the interferon system has been reported, as well as degradation of closely related, but non-identical, mRNAs [14]. A more advanced innovation in functional genomics is now used to enable a more specific determination of genetic expression in the genome.
This is through the use of DNA microarray technology that has paved the way to the accumulation of vast collection of information on genes related to the expression of various diseases. But on the other hand, most of the time the pattern of gene expression is extra complicated to allow identification of the small population of genes not properly expressed that contribute to the causation and maintenance of the disease [14]. Because RNA interference is able to give a relatively convenient ablation of gene expression, it has led to the possibility of using the information on siRNAs to analyse the significance of the pool of thousands of genes.
These can also be used as screening tools for future research studies and medical procedures [16]. Another proposed method is the use of the pool of RNAi viral vectors to apply a selective pressure that will only affect cells that are desired to change can survive. Genes knocked down in the surviving cells can then be identified by sequencing the RNA interference vectors that they carry. This is actually currently used in investigative studies on genes involved in neurodegenerative diseases, diabetes, and cancer [16]. Conclusion
Gene silencing is rapidly become the wave of future genetic and medical discoveries. Its breakthrough discovery has led to dramatic impacts on the human knowledge on how to regulate gene expression, which has a tremendous implication disease treatment and drug development. Especially in the field of HIV and AIDS research, this method is a very promising contributory to the eradication of this presently incurable disease. Although this is relatively new and still has more areas to be discovered, it serendipitous discovery is the hope of future generations towards a life of better quality and longevity.
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