Abstract
Chronic myeloid leukaemia (CML) was one of the first cancers to be linked to a chromosomal abnormality, the Philadelphia chromosome. This chromosome results in a translocation between chromosomes 9 and 22, where the ABL gene on chromosome 9, a tyrosine kinase, is translocated to the BCR gene region on chromosome 22 giving rise to an abnormal BCR/ABL fusion gene. The resultantfusion gene has an abnormally upregulated tyrosine kinase activity that results in an increase in the proliferation of immature white blood cells, thus leading to the development of CML. There are several breakpoints that can occur in the BCR gene two of which give rise to 95% of CML cases.
These fusion points are called the β3α2 and β2α2 depending on where the chromosomal breakages occur in the BCR gene. The aim of the project was to establish a new method of treatment for CML through the use of RNAi to abolish the increase in tyrosine kinase activity of the abnormal fusion gene product.
The K562 and KCL22 cell lines incorporating the β3α2 and β2α2 fusion points respectively were used in this project. The fusion points were cloned and sequenced. The human U6 and H1 promoters, were selected for the production of the antisense molecules and were obtained by PCR of K562 cDNA. Short hairpin RNA molecules were designed to the sequences of the β3α2 and the β2α2 fusion points. These designed shRNA molecules were synthesized as
oligonucleotides and were incorporated into a reverse PCR primer. Cassettes containing shRNA molecules and a respective promoter were produced by means of PCR and the products cloned into pB12mcs-‐eGFP vector, which was used as a GFP reporter system. Constructs were then transfected into the appropriate cell lines, and expression studies including qPCR and Western blot analysis
were conducted, to examine the effects of the designed shRNA constructs to their target sites on both mRNA and protein levels. In addition, these experiments also indicate the efficiency of the construct and also their specificity to their targets.
qPCR and Western blot analysis, show that both the shRNA molecules designed against the β3α2 and the β2α2 fusion points, efficiently induced RNAi based gene silencing to their target sites. In addition, the designed constructs show high specificity to only its target sites and not to other unrelated or related genes. These results point towards the use of molecular modulation of gene expression as a promising strategy for potential CML therapy.
| Date of Award | 2012 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Peter Eagles (Supervisor) & Mark Sanderson (Supervisor) |