The development of widely used genetically modified immune cells is aimed at the treatment of malignant diseases including B cell cancers. For these purposes, T lymphocytes, which are among hard-to-transfect cells, are modified to express a chimeric antigen receptor or CAR. One of the methods showing success in transfecting these cells is electrogenic transfer by electroporation, whereby the vectors used so far, such as pDNA and retroviral vectors, are replaced by mRNA, which is less toxic. In the master's thesis, we tried to achieve the modification of immune cells with mRNA transfer by electroporation. Firstly, both plasmids (with CAR and GFP) were prepared for transcription by PCR to linearize and amplify the desired fragment. Using a commercial kit, DNA was transcribed in vitro and equipped with appropriate stability constructs (5'cap and 3'poly(A) tail) and purified. Selected cells were electroporated with the Neon Transfection System microporator in a combination of two different conditions and five different mRNA quantities. We found that it is possible to express selected molecular constructs by electroporation of mRNA in Jurkat cells, but we did not achieve the expected level of expression success. We conclude that in in general, to achieve high transformation efficiency and survival of electroporated cells before the experiment, additional optimization of electroporation is necessary, despite the use of conditions listed in the literature. The preparation and quality of mRNA vectors should be optimized.
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