Using sodium bisulphite treatment and PCR to construct mammalian anti-HIV-1 long hairpin RNA expression cassettes

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dc.contributor.advisor Dr. Marco Weinberg; Mr. Lee Alagiozoglou en_US
dc.contributor.author Lugongolo, Masixole Yvonne
dc.date.accessioned 2012-05-03T07:37:47Z
dc.date.available 2012-05-03T07:37:47Z
dc.date.issued 2012-05-03
dc.date.submitted 2011
dc.identifier.uri http://hdl.handle.net/10210/4686
dc.description M.Tech. en_US
dc.description.abstract RNA interference (RNAi) is a gene silencing mechanism that uses short RNA duplexes to block gene expression. This mechanism has been widely explored to determine functions of genes. Furthermore, this phenomenon has been used to silence unwanted genes such as viral genes. RNAi has been successfully employed in non-mammalian organisms such as plants, where long dsRNAs (more than 30 bp) have been used without inducing non-specific effects. However, in mammalian cells, cytoplasmic dsRNAs of more than 30 bp trigger non-specific induction of many genes, which may result from the activation of dsRNA-dependent protein kinase (PKR) and 2’,5’-oligoadenylate synthetase (2’,5’-OAS), via the interferon response pathway. In this study, we describe a novel and simple strategy to overcome nonspecific effects induced by longer RNA duplexes. This strategy uses sodium bisulphite which is a mutagen that deaminates cytosine residue to uracil residues in order to introduce mutations in the sense strand of the duplex. Introduction of these mutations results in the formation of G:U pairings between the sense and antisense strands of the long hairpin RNA. RNA duplexes with mismatches have been shown to be able to prevent interferon induction in mammalian cells. According to the obtained results, long hairpins RNA with and without mismatches were unable to inhibit the expression of the target region, which was the U5 region of the HIV-1 subtype C LTR. The U5 region of the LTR is actively involved in the reverse transcription of HIV-1. Therefore silencing of this region would have led to the inhibition or reverse transcription blockage. Furthermore, data showed that the interferon response was induced when using these long hairpin RNA duplexes. Due to the sensitivity of mammalian cells, the action of sodium bisulphite could have stimulated certain genes of the interferon pathway. Even though hairpins constructed in this study were unable to prevent the induction of the interferon response pathway and also could not silence the target, this strategy of using sodium bisulphite has a great potential as shown by its ability to induce changes in cytosine residues and leaving other nucleotides unchanged. en_US
dc.language.iso en en_US
dc.subject RNA editing en_US
dc.subject HIV (Viruses) en_US
dc.subject Polymerase chain reaction en_US
dc.subject Sodium sulfate en_US
dc.subject Small interfering RNA en_US
dc.title Using sodium bisulphite treatment and PCR to construct mammalian anti-HIV-1 long hairpin RNA expression cassettes en_US
dc.type Thesis en_US

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