Rapid breakthrough of the molecular biology allows us simple and efficient synthesis of custom designed recombinant DNA molecules, which can be widely used for studying physiological and pathophysiological molecular mechanisms in the organisms. Proteins from the SOX family are remarkably important transcription factors, that play a significant role in regulation of the gene expression, through enhancer and regulatory regions and they are crucial in the control of cell fate decisions and the ability to differentiate during the embryo development process. Their function in the organism is so important, that potential mutations in the SOX genes, could lead to extremely complex and severe diseases.
Main purpose of the Master's thesis was to synthesize expression plasmid vectors with recombinant DNA technology and successfully clone target coding regions of SOX5 and SOX6 genes. Synthesized recombinant plasmid molecules will, in transfected cell lines, produce tagged fusion SOX5 and SOX6 proteins, which are going to be used in the following studies for additional investigation of their involvement in the molecular mechanisms of gene expression regulation in connection to other diseases development.
We synthesized our recombinant plasmid molecules in the way, that the target coding sequences of the SOX5 and SOX6 genes were precisely inserted into the polyclonal site of pFLAG-CMVTM-2 expression vectors. For cutting plasmids and insert molecules in the process of the convenient molecular cloning, we used NotI and KpnI restriction endonucleases, which generated complementary sticky ends for inserting the target gene coding sequences into cut vector molecules. After ligation, we selected and multiplied recombinant plasmid molecules on the growth medium with ampicillin. At the end, we confirmed molecular cloning with restriction analysis followed by an agarose electrophoresis and Sanger sequencing.
Based on results of confirmatory methods we concluded, that molecular cloning was successful in both cases. Final confirmation was conducted by bioinformatic sequence analysis of the recombinant plasmids, where we proved approximately 99 % matching with applicable reference variants of the SOX5 and SOX6 gene coding sequences.
Due to their versatility and involvement in many physiological and pathophysiological processes, transcription factors from the SOX family, are suitable for further research, because better understanding of their molecular mechanisms could lead us to discovery of new disease markers, therapeutic targets and innovative drugs.
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