Preparation of cDNA of selected gene and cloning.

• Main Author: Bogdanovič, Igor
• Other Authors: Miletín, Miroslav
• Format: Dissertation
• Language: English
• Published: 2011
• Online Access: http://www.nusl.cz/ntk/nusl-298033

ABC drug efflux transporters are transmembrane proteins which are known to play a crucial role in drug resistance. The ability of these proteins to actively transport a wide variety of compounds out of the cell is one of the key reasons behind the resistance of cancer cells to chemotherapeutic agents. To study the activity of efflux transporters and identify their substrates or inhibitors a number of in vitro models have been developed. These models are based on the mammalian cells genetically modified in order to express the protein of interest. For genetic manipulations of the cells various expression plasmids are used. In our study we attempted to prepare a universal expression plasmid, containing a short sequence of V5 tag. Such plasmid enables further cloning of coding sequences of selected efflux transporters so that the V5 tag is localized on their 3'-end. Once constructed, the plasmid can further be used to transfect the desired cell line, thus developing an in vitro cell model. Since the transporter is produced as a fusion protein with the V5 tag localized on their C-end, the tag could be used as a universal marker of the transporter expression detectable by immunohistochemical methods. To construct the V5 tag sequence two approaches were employed both leading to the product of the desired...