Advanced methodsMethods stated below serve to list core capabilities of research department of GB based on our modern equipment. GB offers the methods for two principal parties. The former are those looking for potential scientific co-operation in various projects where we can utilize our equipment, tricky protocols and last but not least, experienced staff. In this field, we will try to meet any partner´s requirement and any form of scientific co-operation is possible. The latter are pharma companies or contract research companies outsorcing part of their non-clinical developmental research. We offer our sponsor capabilities and services outlined below. In vitro test of electrophoretic mobility is used to assess a relevant dissociation constant. Duplex DNA (containing a target sequence for a specific TFO) carrying fluorescent labels is incubated with the TFO under standard triplex-forming conditions. Resulting structure is then subjected to electrophoretic separation on a native vertical polyacrylamide gel. DMS "footprinting" Binding of a TFO to a duplex target (the dsDNA is fluorescent labeled) shelters the N7 position of guanosine residues in the duplex and therefore the arising triplex is protected from reaction with dimethyl sulphate (DMS). By contrast, all guanosine residues outside the triplex zone are modified as usual. Consequently (after the application of piperidin), a characteristic pattern, a so-called DMS footprint, should be seen that could be taken as being diagnostic of triple helix formation. The fragments produced by reaction with DMS will be separated using a DNA capillary array system. DNase I “footprinting” is also used to assess triplex formation. This method is analogous to the DMS technique except that DNase I is employed in lieu of an electrophilic reagent. Study of the melting curves of formed complexes (triplexes) Hyperchromicity effect of DNA is used to study the formation of TFO complexes with target duplex as dependence of absorbance change on temperature. Capillary HPLC columns
Sophisticated cloning experiments The fast success in most cloning experiments usually depends on a reliable experiment design. GB has long time experience in cloning experimental design and research applications. The steps in the cloning experiments usually include the carefull design of the whole cloning procedure, selection of appropriate competent E.coli genotype strain, restrictions, ligations, sequencing and finally desired plasmid(s) isolation and purification in a large scale. We can offer the clone deposition in our E.coli clone deposition and/or DNA clone deposition for faster production of desired plasmid(s) in the future. Transient or stable gene transfection GB has worked out powerful pipeline for several cell-lines non-liposomal transfection, marker gene selection and subsequent clone selection, analysis and isolation. Close linked to the cloning capabilities, GB is able to offer fast corrective reactions to the emerged problems. Our experienced scientists can analyze the problem and suggest the best solution. Retroviral gene delivery and expression The retroviral gene delivery is one of the most powerful techniques for making recombinant eucaryotic cells. A stable transgene integrates into the genome of target cell and is thus inherited in subsequent populations. We utilize several retroviral backbone plasmids with a set of selection or marker genes (e.g. blasticidine, hygromycine or G418 resistance, green fluorescent protein GFP or luciferase). The power of joined core technologies (cloning, cell-lines, transfection and retroviral gene transfer) lies in the easy switch between several types of gene delivery and/or solution of emerging problems. RNA interference In addition to commercial synthetic siRNA synthesis on demand GB has developed a line of recombinant plasmids for generation of shRNA (short hairpin RNA) inside the transfected cells with a set of selection or marker genes on the same plasmid (e.g. hygromycine or G418 resistance, green fluorescent protein GFP or luciferase). In conjunction with our retroviral gene delivery capabilities, selected shRNA expressing plasmids are also available for retroviral infection for stable integration and shRNA production. DNA arrays
Production of recombinant protein GB is capable to develop a protein production based on either baculovirus expression system or mammalian expression system (hCYP1A1, hCYP3A4, hCYPOR, hNSE, hSHBG, hTPO. Eukaryotic production systems are often preferred over prokaryotic ones because of post-translational modification of produced proteins. Although prokaryotic production systems are much simpler and thus cheaper, resulting proteins usually do not meet all the requirements for biologically active protein of good quality. For this reason, GB works primarily with eukaryotic production systems. Production systems used
Protein characterization
Universal cell-based system for testing the nuclear uptake Antisense and antigene triplex forming oligonucleotides are attractive compounds for gene therapy, but major limiting factors for their routine use are inefficient cellular uptake and low accessibility to the target sites. Universal cell-based system is available for monitoring of intracellular and nuclear uptake of oligonucleotides in real time. Cell system is based on Enhanced green fluorescent protein (EGFP) conjugated to Nuclear localization signal (NLS). EGFP-NLS fusion protein is used after the translocation to the nuclei as a positive fluorescence control of cells with active transport paths. Patent no. 295047 - Universal cell-based system for testing the nuclear uptake
Universal cell-based system for triplex forming oligonucleotides function testing Triplex forming oligonucleotides (TFO) as a tool of gene therapy can selectively inhibit gene expression at the transcriptional level or repair genetic defects by direct genome modification in human cells. Universal testing system is available for monitoring of target gene down-regulation after TFO treatment. Cell system is based on stable transfected cell line with luciferase reporter gene and inserted TFO target sequence (TTS). TTS is designed in two variants where corresponding TFO can bind either to the regulation or coding region of the target gene. Patent no. 295463 - Universal cell-based system for triplex forming oligonucleotides function testing Original cell-culture based testing system for potential selective inhibitors of cyclooxygenases (group of drugs) This technology has been recently developed in GB as a result of recently finished Research Centre of Structure and Mechanism of Action of Potential Drugs that was run together with Charles University, Faculty of Pharmacy and pharmaceutical company Zentiva. The system is based on genetically engeneered cell-line, in which just only one isoform of cyclooxygenase is expressed (only COX1 or COX2). Because the cell-lines differ only in the expression of these two genes (and consequent affected genes) we are able to sensitively measure the selective effect of several compounds on the COX1 or COX2 activity. This system is still being upgraded and GB is now preparing similar system that utilises measuring via bioluminiscence.
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