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dc.contributor.authorSrirangalingam, Umasuthan
dc.date.accessioned2015-09-14T16:13:43Z
dc.date.available2015-09-14T16:13:43Z
dc.date.issued2012
dc.identifier.citationSrirangalingam, U. 2012. Modification of splicing with antisense oligonucleotides in the Insulin receptor exon 11 and Apolipoprotein B exon 26. Queen Mary University of London.en_US
dc.identifier.urihttp://qmro.qmul.ac.uk/xmlui/handle/123456789/8650
dc.descriptionPhDen_US
dc.description.abstractBackground - The alternatively spliced insulin receptor (IR) exon 11 (36 nucleotides) and the constitutively spliced Apolipoprotein B (APOB) exon 26 (7572 nucleotides) are examples of the shortest and longest exons in the genome. Aim - The aim of this study was to investigate the regulation of splicing of these 2 exons in cell culture using 2′-O-methyl RNA antisense oligonucleotides (ASOs) and peptide nucleic acid (PNA)-peptide hybrid ASOs. Methods - ASOs were designed to target key sequences involved in the splicing of the IR exon 11 and exonic splicing silencer sequences (ESS) in APOB exon 26. HepG2 cells were reverse-transfected with the ASOs for 48 hours, mRNA harvested and RT-PCR was performed to amplify the IR isoform and APOB cDNAs which were separated by PAGE and quantified. Results Insulin receptor exon 11 - 2′-O-methyl RNA ASOs targeted to two intronic sites, the 3′ half of exon 11 and spanning the entire exon caused significant exon skipping. PNA-peptide hybrids predicted to increase exon 11 splicing, paradoxically caused exon skipping. PNA-peptide hybrids with 3′ tails caused exon 11 skipping more effectively than hybrids with 5′ tails. Apolipoprotein B exon 26 - Only combinations of 2′-O-methyl RNA ASOs targeting multiple ESSs in APOB exon 26 caused a small proportion of aberrant splicing. This consisted of complete exon 26 skipping and the selection of a downstream cryptic 3′ splice site in preference to the native 3′ splice site. Discussion - Exclusion of the IR exon 11 can be induced by targeting a combination of intronic or exonic sequences. PNA-peptide hybrid ASOs were unable to increase exon 11 splicing. The aberrant splicing of large constitutive exons such as APOB exon 26 can be induced by targeting multiple ESS sites along its course.en_US
dc.description.sponsorshipJoan Adams Fellowship; Barts and the London Charity
dc.language.isoenen_US
dc.publisherQueen Mary University of London
dc.subjectPoliticsen_US
dc.subjectThatcherismen_US
dc.subjectneo-liberalismen_US
dc.titleModification of splicing with antisense oligonucleotides in the Insulin receptor exon 11 and Apolipoprotein B exon 26.en_US
dc.typeThesisen_US
dc.rights.holderThe copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author


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