Transcription activity of transposon sequence limits Sleeping Beauty transposition.

Sleeping Beauty (SB) transposon based technology has been extensively applied in basic research and biotechnology for routine cell culture gene delivery and vertebrate transgenesis, and it is also investigated in various gene therapy applications. Cell tolerance for the transgene is a key factor during transgenesis and is modulated not only through the type but by the dose of expression. Our experimental results exemplify that transgenes regulated with high activity promoters can reduce the overall success of gene delivery. Observations connected to transposon donors regulated by different promoters have also revealed inverse correlation between transcription activity and the hyperactive variant SB100X excision efficiency. This competition between transcription and transposition was independent of the transgene coding sequence and did not alter the transgenic efficiency in general. However, promoters applied in the transgene cassette can produce different average copy numbers depending on the transcriptional activity of the transposon. Unlike the piggyBac (PB) transposon system, this phenomenon allows a fine balance of expression using the high copy potential SB system that adjusts the copy number of lower activity promoter driven transgenes to a higher expression level. All this contributes to a well-tolerated and satisfactory transgenesis, and would be important to consider in gene therapy applications

Az ABCG2 multidrog transzporter fehérje szerkezetének és működésének vizsgálata = Structure and function of the multidrug transporter ABCG2

Az ABCG2 multidrog transzporternek fontos szerepe van mind a daganatok kemoterápia-rezisztenciájában, mind a fiziológiás xenobiotikum transzportban. A projektben előállítottuk az ABCG2 fehérje különböző mutáns és polimorf változatait, elvégeztük ezek részletes funkcionális vizsgálatát. Egy sejtfelszínen reagáló, konformáció-érzékeny anti-ABCG2 monoklonális antitest alkalmazásával felderítettük a transzporter funkcionális állapotait, kémiai módosítások és mutációk segítségével elvégeztük az epitópok jellemzését és molekuláris szintű modellezését. Részletesen elemeztük az ABCG2 transzporter és a membrán lipidek kölcsönhatásait, megállapítottuk a membrán koleszterin jelentős szabályozó szerepét. Az ABCG2 transzporter és célzott hatású rákellenes vegyületek kölcsönhatásainak vizsgálata során klinikailag is alkalmazott gyógyszerekre vonatkozóan kaptunk új információkat. Új módszereket fejlesztettünk ki az ABCG2 szabályozásának, lokalizációjának és funkciójának vizsgálatára, elemeztük a transzporter expresszióját humán embrionális őssejtekben. Több, magas impakt faktorú nemzetközi folyóiratban közöltünk a témáról review cikkeket. | The human ABCG2 multidrug transporter plays a key role in the chemotherapy resistance of malignant tumors, as well as in the physiological elimination of xenobiotics. In this project we have prepared and expressed various mutant and polymorphic variants of the transporter, performed their detailed functional characterization. By using a cell-surface reacting, conformation-sensitive monoclonal antibody against ABCG2, we mapped the functional states of the transporter. In these experiments we applied specific chemical modifications and generated site-directed mutations to characterize the extracellular loop epitope region of ABCG2 and constructed a molecular model for this part of the transporter. We have investigated the modulation of ABCG2 by membrane lipids and found a major role for cholesterol in regulating the transport activity of this protein. By examining a number of new targeted anticancer agents we found that ABCG2 interacts with several of these compounds and may be involved in the resistance against clinically applied molecules. We have developed new methods for studying the regulation, localization and function of the ABCG2 protein, examined the expression profile of this transporter in human embryonic stem cells. During this project we have published several review articles in high-impact international journals

Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications

There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the DeltaDeltaCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies

Partial Disturbance of Microprocessor Function in Human Stem Cells Carrying a Heterozygous Mutation in the DGCR8 Gene

Maturation of microRNAs (miRNAs) begins by the “Microprocessor” complex, containing the Drosha endonuclease and its partner protein, "DiGeorge Syndrome Critical Region 8" (DGCR8). Although the main function of the two proteins is to coordinate the first step of precursor miRNAs formation, several studies revealed their miRNA-independent functions in other RNA-related pathways (e.g., in snoRNA decay) or, for the DGCR8, the role in tissue development. To investigate the specific roles of DGCR8 in various cellular pathways, we previously established a human embryonic stem-cell (hESC) line carrying a monoallelic DGCR8 mutation by using the CRISPR-Cas9 system. In this study, we genetically characterized single-cell originated progenies of the cell line and showed that DGCR8 heterozygous mutation results in only a modest effect on the mRNA level but a significant decrease at the protein level. Self-renewal and trilineage differentiation capacity of these hESCs were not affected by the mutation. However, partial disturbance of the Microprocessor function could be revealed in pri-miRNA processing along the human chromosome 19 miRNA cluster in several clones. With all these studies, we can demonstrate that the mutant hESC line is a good model to study not only miRNA-related but also other “noncanonical” functions of the DGCR8 protein

A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers

Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca2+ oscillation and sustained Ca2+ signals at 1muM and10 muM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI