Untersuchungen kombinierter DNA-Protein-Transduktion zur Optimierung nicht-viralen Gentransfers

Zur Etablierung eines rekombinanten phiC31-TAT-Systems für den nicht-viralen Gentransfer wurden verschiedene phiC31-Integrase-Konstrukte in E. coli BL21 Bakterien rekombinant exprimiert, chromatographisch aufgereinigt und sowohl im Reagenzglas als auch in renalen, alveolären und T-Zelllinien auf Funktionalität getestet. Zur Transduktion dieser Integrase-Plasmid-Komplexe in unterschiedliche Zelltypen wurden verschiedene Transduktionsmethoden verwendet. Stellvertretend für Lunge, Niere und Lymphozyten wurden A549-, 293- und Jurkat-T-Zellen mit Komplexen aus rekombinanter Integrase und einem Luziferase-attB-Plasmid behandelt und über einen Zeitraum von einem Monat auf Luziferaseexpression getestet. Der kombinierte phiC31-Integraseprotein-Plasmid-Transfer ist eine vielversprechende Anwendung für den nicht-viralen Gentransfer, was insbesondere durch die Vermittlung stabiler Langzeittransgenexpression in verschiedenen Zelltypen gezeigt werden konnte

Diagnosing and mapping pulmonary emphysema on X-ray projection images

To assess whether grating-based X-ray dark-field imaging can increase the sensitivity of X-ray projection images in the diagnosis of pulmonary emphysema and allow for a more accurate assessment of emphysema distribution. Lungs from three mice with pulmonary emphysema and three healthy mice were imaged ex vivo using a laser-driven compact synchrotron X-ray source. Median signal intensities of transmission (T), dark-field (V) and a combined parameter (normalized scatter) were compared between emphysema and control group. To determine the diagnostic value of each parameter in differentiating between healthy and emphysematous lung tissue, a receiver-operating-characteristic (ROC) curve analysis was performed both on a per-pixel and a per-individual basis. Parametric maps of emphysema distribution were generated using transmission, dark-field and normalized scatter signal and correlated with histopathology. Transmission values relative to water were higher for emphysematous lungs than for control lungs (1.11 vs. 1.06, p<0.001). There was no difference in median dark-field signal intensities between both groups (0.66 vs. 0.66). Median normalized scatter was significantly lower in the emphysematous lungs compared to controls (4.9 vs. 10.8, p<0.001), and was the best parameter for differentiation of healthy vs. emphysematous lung tissue. In a per-pixel analysis, the area under the ROC curve (AUC) for the normalized scatter value was significantly higher than for transmission (0.86 vs. 0.78, p<0.001) and dark-field value (0.86 vs. 0.52, p<0.001) alone. Normalized scatter showed very high sensitivity for a wide range of specificity values (94% sensitivity at 75% specificity). Using the normalized scatter signal to display the regional distribution of emphysema provides color-coded parametric maps, which show the best correlation with histopathology. In a murine model, the complementary information provided by X-ray transmission and dark-field images adds incremental diagnostic value in detecting pulmonary emphysema and visualizing its regional distribution as compared to conventional X-ray projections

Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes

Characterization of Ku702–NLS as Bipartite Nuclear Localization Sequence for Non-Viral Gene Delivery

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku702-NLS), a nuclear transport active mutant of Ku702-NLS (s1Ku702-NLS) and a nuclear transport deficient mutant of Ku702-NLS (s2Ku702). We examined the transfection efficiency of binary Ku702-NLS/DNA and ternary Ku702-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku702-NLS and s1Ku702-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer

Drosophila CP190- and dCTCF-mediated enhancer blocking is augmented by SUMOylation

Abstract Background Chromatin insulators shield promoters and chromatin domains from neighboring enhancers or chromatin regions with opposing activities. Insulator-binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the small ubiquitin-like modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes. Results Here we addressed the impact of dSUMO in respect of insulator function, chromatin binding of insulator factors and formation of insulator speckles in Drosophila. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190. Conclusions These results indicate that enhanced chromatin binding of SUMOylated CP190 causes fusion of insulator speckles, which may allow for more efficient insulation

Analysing luciferase expression in lung homogenisates after nasal instillation of gene transfer agents.

<p>1, Ku70₂-NLS/PEI/DNA; 2, s1Ku70₂-NLS/PEI/DNA; 3, s2Ku70₂/PEI/DNA; 4, PEI/DNA. Gene vector complexes were formulated as follows: 30 µg DNA; NLS: ± ratio = 5; PEI: N/P ratio = 10. Luciferase activity was measured in lung homogenisates 24 h after application of gene vectors. Values between Ku70₂-NLS/PEI and PEI significantly different (<i>p</i>≤0.043; n = 4). Statistical analysis was performed using the Mann-Whithney-U test.</p

Ku70₂-NLS in comparison to monopartite nuclear localization sequences.

<p>BEAS-2B cells were transfected with Ku70₂-NLS/DNA complexes or the monopartite active NLS (TAT)₂ or NLSV404, and their nuclear transport deficient sequences (control) NLS (TAT2M1 or cNLS). All the complexes were generated in HBS (charge ± = 5). Transgene expression of Ku70₂-NLS and s1Ku70₂-NLS was significantly higher (p<0.05, Mann-Whitney-U-Test) compared to the respective monopartite NLS (TAT)₂ or NLSV404. Transgene expression of Ku70₂-NLS and s1Ku70₂-NLS compared to the control peptide s2Ku70₂ was close to significance. This study clearly suggests an advantage of the bipartite NLS over monopartite NLS.</p

Transfection efficiency of ternary gene vector complexes using transMag^PEI and different solvents.

<p>BEAS-2B cells were transfected with gene vector complexes. Transgene expression of Ku70₂-NLS/transMag^Pei/DNA is more efficient than s2Ku70₂/transMag^Pei and transMag^Pei/DNA. *p = 0.02 and **p = 0.001.</p

Intracellular localization of β-galactosidase fusion proteins.

<p>BEAS-2B cells were electroporated with 10 µg β-galactosidase coding plasmid DNA. All images show the typical blue β-galactosidase staining. Images (a) and (b) point that Ku70₂-NLS and s1Ku70₂-NLS show nuclear localization signal activity. The exclusive blue staining of nuclei is clearly visible. The s2Ku70₂ shows staining of the cytosol. The NLS large antigen (e) was used as positive control for nuclear localisation and β-galactosidase without NLS (d) as negative control.</p

MOESM1 of Drosophila CP190- and dCTCF-mediated enhancer blocking is augmented by SUMOylation

Additional file 1: Figure S1. dCTCF and CP190 peaks co-localize with previously identified dCTCF and CP190 binding sites. Peaks for dCTCF and CP190 were identified using MACS2 based on publicly available (GSE41354) ChIP-seq profiles published in Ong et al. [42] (PMID 24055367). Average binding of dCTCF as well as CP190 before and after expression of FLAG-dSUMO is shown across the known dCTCF (left) and CP190 (right) binding sites