| Summary: | Cystic fibrosis is a genetic disease caused by a mutation in the CFTR gene. This leads to an absence or a malfunction of CFTR chloride channel, which is also a crutial regulator of other ion channels. This thesis was aimed at gene therapy of cystic fibrosis using CFTR- mRNA gene transfer. To determine the expression of CFTR protein, methods of indirect immunofluorescence and Western blot immunodetection were utilized. Also relative gene expression levels of CFTR gene were assessed by quantitative PCR. Experiments were carried out on a healthy lung epithelial cell line (NuLi-1), a lung epithelial cell line with F508del mutation (CuFi-1) and an embryonic kidney epithelial cell line (HEK293S). CFTR protein was visualized by previously mentioned methods using six primary antiobodies (432, 450, 570, 596, 769, CF3). Primary antibodies 570 a CF3 were found as optimal for the detection of CFTR protein by the method of indirect immunofluorescence, whereas for the detection of this protein by the method of Western blot only the CF3 antibody was suitable. It was also determined that CFTR gene is expressed in overall small levels. Its relative gene expression in the CuFi-1 cell line was approximately six times higher than in the NuLi-1 cell line. The efficiency of transfection of CuFi-1 cell line by CFTR-mRNA...
|
|---|
