Cloning and expression of wild-type and mutated forms of Bcr-Abl in a mouse pro-B cell line
AuthorHammer, Stine Gangnæs
Chronic myelogenous leukaemia is a monoclonal hematopoetic stem cell disorder characterised by the t(9;22) translocation and results in the constitutively activated Bcr-Abl tyrosine kinase. Since the tyrosine kinase activity of the Bcr-Abl fusion protein is the causative molecular event in CML, targeting the tyrosine kinase activity appears to be an attractive therapeutic strategy. Imatinib, Glivec, is a drug that inhibits the tyrosine kinase activity of Bcr-Abl. By binding to the ATP binding pocket, it prevents ATP from binding and the phosphorylation of downstream substrates is disrupted. Clinical studies have proven imatinib to be highly effective in the treatment of CML and imatinib is now the first-line therapy for all stages of CML However; point mutations have been detected in the ATP binding region of the Abl kinase domain. These mutations alter the conformation of the ATP binding pocket, disturb the binding of imatinib, and lead to imatinib resistance. We wanted to develop an experimental system where the effects of mutations in Bcr-Abl, leading to imatinib resistance, could be studied and new targets for therapy identified. For this we were going to clone Bcr-Abl into a pMACS 4-IRES.II vector. The Bcr-Abl gene is large, so to get the full-length construct, the cloning strategy involved ligation of PCR fragments in a stepwise order. Once inside the vector, the construct had to be transfected into BA/F3 cells. To study single point mutations some of the relevant point mutations were supposed to be subcloned into the Bcr-Abl construct and expressed in BA/F3 cells. To monitor the transfection and selection strategy with the pMACS 4-IRES.II vector and the BA/F3 cells, a pilot study was performed. A GFP gene was cloned into the pMACS 4-IRES.II vector and transfected into the BA/F3 cells. Expressed GFP will make fluorescent light that can be observed in a microscope. In conclusion, the cloning of this long Bcr-Abl gene proved to be more difficult than expected. First, misannealing resulted in an incomplete PCR product, which forced us to develop another strategy for this fragment. The 5’ part and the 3’part of Bcr-Abl was then successfully cloned in two vectors. However, all attempts to try to join the different Bcr-Abl fragments into one vector failed.
PublisherUniversitetet i Tromsø
University of Tromsø
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