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Possibel approaches to gene therapy of cystic fibrosis
Král, Jan ; Bořek Dohalská, Lucie (advisor) ; Vaněk, Ondřej (referee)
1 Abstract Cystic fibrosis is an autosomal recesive disorder caused by a mutation in the CFTR gene, which leads to inefficiency or absence of CFTR chlorid channel. One way to induce production of CFTR protein in target cells, is to use gene therapy. The principle od gene therapy is to transfer DNA or mRNA molecules inside malfunctioning cells. The aim of this study was to optimise the detection of CFTR protein using the Western blot analysis. Then, using this method the effectiveness of CFTR-mRNA transfection was studied. To study the CFTR protein, a number of cell lines was used: a healthy human ephitelial cell line (NuLi-1), an ephitelial cell line with ΔF508 mutation (CuFi-1), and a human lung carcinoma cell line (A549). This study compared four different ways of cell lysis - lysis by sonication and lysis by three distinct lysis buffers. Lysis by RIPA buffer with protease inhibitors was determined for the detection of CFTR protein. Moreover, three different primary monoclonal antibodies were also tested. The CF3 antibody, which is specific to an extracellular epitope of CFTR protein, was found able to detect CFTR protein specifically. A couple of different glycosylated forms of CFTR protein was detected. The highest amount of CFTR protein was determined in the NuLi-1 cell line. CFTR protein was also...
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