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National Repository of Grey Literature 2 records found  Search took 0.00 seconds. 
Preparation of lentiviral expression vector with reporter gene
 Skořepa, Ondřej ; Vaněk, Ondřej (advisor) ; Milichovský, Jan (referee)
Besides recombinant protein expression in prokaryotic cell lines (E. coli), systems, that could quickly, reliably and stably produce recombinant proteins in human cell lines, come to the fore. These cell lines assure proper tertiary structure and post-translational modification of the desired products. One of the ways to achieve production of recombinant proteins in human cell lines is the use of lentiviral vectors. This thesis describes the preparation of the lentiviral vector (plasmid) Daedalus, which contains a construct for recombinant expression of secreted alkaline phosphatase. For the preparation of the desired plasmid methods based on insertion of the secreted alkaline phosphatase gene using the restriction endonucleases and methods based on amplification by polymerase chain reaction (restriction-free cloning, transfer polymerase chain reaction and Gibson assembly) were used.
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Optimization of Culture Medium for HEK293 Cell Line
 Čuperková, Romana ; Vaněk, Ondřej (advisor) ; Šácha, Pavel (referee)
HEK293 is a human cell line derived from embryonic kidney cells and is a frequently used system for the production of recombinant proteins. This work dealt with optimization of the composition of serum-free medium for HEK293S and HEK293T cell lines as a compensation for expensive commercial media. The growth of culture and expression of reporter proteins SEAP and GFP was monitored as the markers. I managed to create a new medium which contained, among other compounds, insulin (1 mg/l), transferrin (5 mg/l) and a mixture of trace elements. During the cultivation in a mixture of commercial medium EX CELL 293 with my new medium 293S cells grew faster than during the cultivation in commercial media (doubling time 20,47 ± 2,68 hours (srel = 13,1 %)). It seems that the new medium is suitable for transfection of HEK293 cell lines with a relatively high expression of recombinant proteins. Transfection ratio of DNA:PEI (w/w) for this medium is 1:2 to 1:3.
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