Podcasts about egfp

BUFFALO, NY- June 17, 2024 – A new research paper was published on the cover of Aging (listed by MEDLINE/PubMed as "Aging (Albany NY)" and "Aging-US" by Web of Science) Volume 16, Issue 11, entitled, “Mitophagy and cancer: role of BNIP3/BNIP3L as energetic drivers of stemness features, ATP production, proliferation, and cell migration.” Mitophagy is a selective form of autophagy which permits the removal of dysfunctional or excess mitochondria. This occurs as an adaptative response to physiological stressors, such as hypoxia, nutrient deprivation, or DNA damage. Mitophagy is promoted by specific mitochondrial outer membrane receptors, among which are BNIP3 and BNIP3L. The role of mitophagy in cancer is being widely studied, and more specifically in the maintenance of cancer stem cell (CSC) properties, such as self-renewal. Given that CSCs are responsible for treatment failure and metastatic capacity, targeting mitophagy could be an interesting approach for CSC elimination. In this new study, researchers Marta Mauro-Lizcano, Federica Sotgia, and Michael P. Lisanti from the University of Salford describe a new model system to enrich sub-populations of cancer cells with high basal levels of mitophagy, based on the functional transcriptional activity of BNIP3 and BNIP3L. “Briefly, we employed a BNIP3(L)-promoter-eGFP-reporter system to isolate cancer cells with high BNIP3/BNIP3L transcriptional activity by flow cytometry (FACS).” The model was validated by using complementary lysosomal and mitophagy-specific probes, as well as the mitochondrially-targeted red fluorescent protein (RFP), namely mt-Keima. High BNIP3/BNIP3L transcriptional activity was accompanied by increases in i) BNIP3/BNIP3L protein levels, ii) lysosomal mass, and iii) basal mitophagy activity. Furthermore, cancer cells with increased BNIP3/BNIP3L transcriptional activity exhibited CSC features, such as greater mammosphere-forming ability and high CD44 levels. “To further explore the model, we also analysed other stemness characteristics in MCF7 and MDA-MB-231 breast cancer cell lines, directly demonstrating that BNIP3(L)-high cells were more metabolically active, proliferative, migratory, and drug-resistant, with elevated anti-oxidant capacity. Therefore, high levels of basal mitophagy appear to enhance CSC features.” DOI - https://doi.org/10.18632/aging.205939 Corresponding authors - Federica Sotgia - fsotgia@gmail.com, and Michael P. Lisanti - michaelp.lisanti@gmail.com Video short - https://www.youtube.com/watch?v=n872jCkc-q8 Sign up for free Altmetric alerts about this article - https://aging.altmetric.com/details/email_updates?id=10.18632%2Faging.205939 Subscribe for free publication alerts from Aging - https://www.aging-us.com/subscribe-to-toc-alerts About Aging-US Aging publishes research papers in all fields of aging research, including but not limited to aging processes (from yeast to mammals), cellular senescence, age-related diseases (such as cancer and Alzheimer's disease) and their prevention and treatment, anti-aging strategies and drug development, and, importantly, the role of signal transduction pathways in aging (such as mTOR) and potential approaches to modulate these signaling pathways to extend lifespan. The journal aims to promote 1) treatment of age-related diseases by slowing down aging, 2) validation of anti-aging drugs by treating age-related diseases, and 3) prevention of cancer by inhibiting aging. (Cancer and COVID-19 are age-related diseases.) Please visit our website at https://www.Aging-US.com​​ and connect with us: Facebook - https://www.facebook.com/AgingUS/ X - https://twitter.com/AgingJrnl Instagram - https://www.instagram.com/agingjrnl/ YouTube - https://www.youtube.com/@AgingJournal LinkedIn - https://www.linkedin.com/company/aging/ Pinterest - https://www.pinterest.com/AgingUS/ Spotify - https://open.spotify.com/show/1X4HQQgegjReaf6Mozn6Mc MEDIA@IMPACTJOURNALS.COM

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.23.550233v1?rss=1 Authors: Ikawa, K., Hiro, S., Kondo, S., Ohsawa, S., Sugimura, K. Abstract: Directional cell rearrangement is a critical process underlying correct tissue deformation during morphogenesis. Although the involvement of F-actin regulation in cell rearrangement has been established, the role and regulation of actin binding proteins (ABPs) in this process are not well understood. In this study, we investigated the function of Coronin-1, a WD-repeat actin-binding protein, in controlling directional cell rearrangement in the Drosophila pupal wing. Transgenic flies expressing Coronin-1-EGFP were generated using CRISPR-Cas9. We observed that Coronin-1 localizes at the reconnecting junction during cell rearrangement, which is dependent on actin interacting protein 1 (AIP1), an actin disassembler and a known regulator of wing cell rearrangement. Loss of Coronin-1 function reduces cell rearrangement directionality and hexagonal cell fraction. These results suggest that Coronin-1 promotes directional cell rearrangement via its interaction with AIP1, highlighting the role of ABPs in the complex process of morphogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.14.549007v1?rss=1 Authors: Niu, C., Yue, X., An, J. J., Xu, H., Xu, B. Abstract: The brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase receptor B (TrkB) are widely expressed in the central nervous system. It is well documented that neurons express BDNF and full-length TrkB (TrkB.FL), and a lower level of truncated TrkB (TrkB.T). With conflicting results, glial cells also have been reported to express BDNF and TrkB. In the current study, we employed a more sensitive and reliable genetic method to characterize the expression of BDNF and TrkB in glial cells in the mouse brain. We utilized three Cre mouse strains in which Cre recombinase is expressed in the same cells as BDNF, TrkB.FL, or all TrkB isoforms, and crossed them to Cre-dependent EGFP reporter mice to label BDNF- or TrkB- expressing cells. We performed immunohistochemistry with glial cell markers to examine the expression of BDNF and TrkB in microglia, astrocytes, and oligodendrocytes. Surprisingly, we found no BDNF- or TrkB- expressing microglia in the brain and spinal cord. Consistent with previous studies, most astrocytes only express TrkB.T in the adult brain. Moreover, there are a small number of astrocytes and oligodendrocytes that express BDNF, the function of which is to be determined. We also found that oligodendrocyte precursor cells, but not mature oligodendrocytes, express both TrkB.FL and TrkB.T in the adult brain. These results not only clarify the expression of BDNF and TrkB in glial cells, but also open opportunities to investigate previously unidentified roles of BDNF and TrkB in glial cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.04.527050v1?rss=1 Authors: Yu, V., Yong, F., Chen, K., Georgiadou, E., Parveen, N., Marta, A., Khadayate, S., Stamoulis, Z., Marselli, L., De Luca, C., Suleiman, M., Hatanaka, Y., Montoya, A., Elliott, J., Patel, B., Demchenko, N., Whilding, C., Hajkova, P., Schliaha, P., Kramer, H., Ali, Y., Marchetti, P., Dhawan, S., Withers, D. J., Rutter, G. A., Millership, S. J. Abstract: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. The molecular mechanisms through which beta cell hierarchy is established remain poorly understood. The neuronatin (Nnat) gene is imprinted in mice and humans and is required for normal insulin synthesis and secretion. Here, we demonstrate that Nnat is differentially expressed in a discrete beta cell population in a developmental-stage and DNA methylation (DNMT3A)-dependent manner. Explored in mice expressing eGFP from a bacterial artificial chromosome-expressed transgene, NNAT+ cells displayed a discrete transcriptome, and appear to represent a beta cell population specialised for insulin production. Correspondingly, highly connected hub cells were less abundant in the NNAT+ population. These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.01.526373v1?rss=1 Authors: Tian, T., Ward, P. J. Abstract: The global double-fluorescent cre-dependent reporter mouse mT/mG has been a valuable tool for differential identification of tissues and cells. This line was previously shown to have EGFP expression in a cre-dependent fashion in single neurons. Here, we demonstrate very sparse labeling in a ThCre:mT/mG cross. This is evident in sweat glands, thoracic spinal cord, sciatic nerve, and lumbar sympathetic ganglia. Therefore, this model may be suitable for imaging or electrophysiology studies of postganglionic sympathetic neuron cell bodies but ineffective for visualizing peripheral axons and tissue targets of the sympathetic nervous system. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.27.525833v1?rss=1 Authors: Gonzales, D. T., Schuhmacher, M., Lennartz, H. M., Iglesias-Artola, J. M., Kuhn, S. M., Barahtjan, P., Zechner, C., Nadler, A. Abstract: Lipids are key components of cellular signaling networks yet studying the role of molecularly distinct lipid species remains challenging due to the complexity of the cellular lipidome and a scarcity of methods for performing quantitative lipid biochemistry in living cells. We have recently used lipid uncaging to quantify lipid-protein affinities and rates of lipid transbilayer movement and turnover in the diacylglycerol cascade using population average time series data. So far, this approach does not allow to account for the cell-to-cell variability of cellular signaling responses. We here aim to develop a framework that allows to quantitatively determine diacylglycerol-protein affinities and transbilayer movement at the single cell level. A key challenge is that initial uncaging photoreaction yields cannot be measured for single cells and have to be inferred along with the remaining model parameters. We first performed an in silico study on simulated data to understand under which conditions all model parameters are well identifiable. Using profile likelihood analysis, we found that identifiability depends predominantly on the signal-to-noise ratio. The impaired parameter identifiability due to experimental noise can be partially mitigated by increasing the number of single cell trajectories. Using a C1-domain-EGFP fusion protein as a model effector protein in combination with a broad variety of structurally different diacylglycerol species, we acquired multiple sets of single cell signaling trajectories. Using our analytical pipeline, we found that almost all species-specific model parameters are identifiable from experimental data. We find that higher unsaturation degree and longer side chains correlate with faster lipid transbilayer movement and turnover and higher lipid-protein affinities, with the exception of steaoryl-oleoyl glycerol, which noticeably deviated from the general trend. In summary, our work demonstrates how rate parameters and lipid-protein affinities can be quantified from single cell signaling trajectories with sufficient sensitivity to resolve the subtle kinetic differences caused by the chemical diversity of signaling lipid pools. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.06.519168v1?rss=1 Authors: Benitez, C., Stephensen, H. J., Benraiss, A., Mokso, R., Sporring, J., Goldman, S. Abstract: Astroglial dysfunction contributes to the pathogenesis of Huntington's disease (HD), and glial replacement can ameliorate disease course. To establish the topographic relationship of diseased astrocytes to medium spiny neuron (MSN) synapses in HD, we used 2-photon imaging to map the relationship of tRFP-tagged striatal astrocytes and rabies-traced, EGFP-tagged coupled neuronal pairs, in R6/2 HD and wild-type (WT) mice. The tagged, prospectively-identified corticostriatal synapses were then studied by correlated light electron microscopy followed by serial block-face scanning EM, allowing nm scale assessment of synaptic structure in 3D. By this means, we compared the astrocytic engagement of single striatal synapses in HD and WT brains. R6/2 HD astrocytes exhibited constricted domains, with significantly less coverage of mature dendritic spines than WT astrocytes, despite enhanced engagement of immature, thin spines. These data suggest that disease-dependent changes in astroglial engagement and sequestration of MSN synapses enable the high synaptic and extrasynaptic levels of glutamate and K+ that underlie the striatal hyperexcitability of HD. As such, these data suggest that astrocytic structural pathology may causally contribute to the synaptic dysfunction and disease phenotype of those neurodegenerative disorders characterized by network overexcitation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.24.517872v1?rss=1 Authors: Mamun, M. A. A., Reza, M. A., Islam, M. S. Abstract: Lipid droplets (LDs) are storage organelles for neutral lipids. Our knowledge about fungal LD biogenesis is limited to budding yeast. Moreover, regulation of LD in multinucleated filamentous fungi with considerable metabolic activity is unknown. Here, 19 LD-associated proteins were identified in Aspergillus oryzae using colocalization screening of a previously established Enhanced green fluorescent protein (EGFP) fusion proteins library. Following a functional screening, 12 candidates have been identified as lipid droplet regulating (LDR) proteins, the loss of which resulted in aberrant LD biogenesis. LDR proteins bind to LD via the insertion of the putative amphipathic helices (AHs) which were investigated using AH-disruptive mutations and subsequent imaging. Further analysis revealed that LdrA with Opi1 domain is essential for cytoplasmic and nuclear LD biogenesis via a novel AH. Phylogenetic analysis demonstrated the pattern of their evolution, which was predominantly based-on gene duplication. Our study provides substantial molecular insights into LD biogenesis and creates a breakthrough in using reverse genetics for identifying LD-regulating proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.04.515095v1?rss=1 Authors: Sun, T., Grassam-Rowe, A., Pu, Z., Ren, H., An, Y., Guo, X., Hu, W., Liu, Y., Li, Y., Liu, Z., Kou, K., Ou, X., Chen, T., Fan, X., Liu, Y., Tu, S., He, Y., Ren, Y., Chen, A., Shang, Z., Xia, Z., Miquerol, L., Smart, N., Zhang, H., Tan, X., Shou, W., Lei, M. Abstract: Cardiac conduction system (CCS) morphogenesis is essential for correct heart function yet is incompletely understood. Here we established the transcriptional landscape of cell types populating the developing heart by integrating single-cell RNA sequencing and spatial enhanced resolution omics-sequencing (Stereo-seq). Stereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. This led to the identification of a previously unrecognized cardiomyocyte population expressing dopamine beta-hydroxylase (Dbh+-CMs), which is closely associated with the CCS in transcriptomic analyses. To confirm this finding, genetic fate mapping by using DbhCre/Rosa26-tdTomato reporter mouse line was performed with Stereo-seq, RNAscope, and immunohistology. We revealed that Dbh+-derived CMs first emerged in the sinus venosus at E12.5, then populated the atrial and ventricular CCS components at E14.5, with increasing abundance towards perinatal stages. Further tracing by using DbhCFP reporter and DbhCreERT/Rosa26-tdTomato inducible reporter, we confirmed that Dbh+-CMs are mostly abundant in the AVN and ventricular CCS and this persists in the adult heart. By using DbhCre/Rosa26-tdTomato/Cx40-eGFP compound reporter line, we validated a clear co-localization of tdTomato and eGFP signals in both left and right ventricular Purkinje fibre networks. Finally, electrophysiological optogenetic study using cell-type specific Channelrhodopsin2 (ChR2) expression further elucidated that Dbh+-derived CMs form a functional part of the ventricular CCS and display similar photostimulation-induced electrophysiological characteristics to Cx40CreERT/ChR2- tdTomato CCS components. Thus, by utilizing advanced transcriptomic, mouse genetic, and optogenetic functional analyses, our study provides new insights into mammalian CCS development and heterogeneity by revealing novel Dbh+-CMs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Segunda mesa del Especial Pensiones en Capital Intereconomía. Mª Eugenia Líbano, responsable Formación Comercial de Santa Lucía Vida y Pensiones; Carolina Mateo Gracia, Jefa de Desarrollo de Negocio Ibercaja Pensión EGFP; Ignasi Viladesau (Director de Inversiones de MYINVESTOR) y Juan Jesús Gómez Gómez, director de Previsión (Seguros de Ahorro y Pensiones) de Banco Sabadell debaten entre otras cuestiones sobre los vehículos de inversión que nos pueden ayudar a ahorrar pensando en la jubilación.

Gonzalo Sánchez, gestor de Renta Variable Iberia de Gesconsult, ha analizado en Capital Intereconomía la situación actual de los mercados. Considera que el fondo de mercado «todavía es bueno gracias a los mensajes de los bancos centrales» que están quitando importancia al aumento de la inflación y eso provoca que los inversores entiendan que no se van a retirar los estímulos de manera anticipada. ¿Qué buscan los clientes de IronIA Fintech?. Nos lo explica su Ceo, José Antonio Esteban. Consultorio de bolsa con Miguel Méndez, Director General de Metagestión. ¿Es buen momento para los sectores cíclicos?. Lo comentamos en el foro de la inversión con Mario González y Álvaro Fernández, directores de desarrollo de negocio de Capital Group para Iberia. Segunda mesa del Especial Pensiones en Capital Intereconomía. Mª Eugenia Líbano, responsable Formación Comercial de Santa Lucía Vida y Pensiones; Carolina Mateo Gracia, Jefa de Desarrollo de Negocio Ibercaja Pensión EGFP; Ignasi Viladesau (Director de Inversiones de MYINVESTOR) y Juan Jesús Gómez Gómez, director de Previsión (Seguros de Ahorro y Pensiones) de Banco Sabadell debaten entre otras cuestiones sobre los vehículos de inversión que nos pueden ayudar a ahorrar pensando en la jubilación.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.374231v1?rss=1 Authors: Blumenstock, S., Schulz-Trieglaff, E. K., Bolender, A.-L., Voelkl, K., Lapios, P., Lindner, J., Hipp, M. S., Hartl, U., Klein, R., Dudanova, I. Abstract: The cellular protein quality control machinery is important for preventing protein misfolding and aggregation, and decline in protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how proteostasis capacity of neurons changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc), a conformationally unstable protein that requires chaperones for proper folding and sensitively reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked impairment in proteostasis in tauopathy mice, but not in Huntington's disease mice. Mechanistic investigations in primary neuronal cultures demonstrate that cytoplasmic, but not nuclear, aggregates cause defects of cellular protein quality control. Thus, the Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.27.357418v1?rss=1 Authors: Cihlova, B., Huskova, A., Böserle, J., Nencka, R., Boura, E., Silhan, J. Abstract: Spanish flu and other influenza outbreaks, the recent Zika epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases that are caused by RNA viruses. Perhaps less well-known yet dangerous RNA viruses cause deadly diseases such as polio, Ebola, measles, rubella, yellow fever, dengue fever, etc. To combat a particular viral disease by diminishing its spread and number of fatal cases, effective vaccines and antivirals are indispensable. Therefore, quick access to the means of discovery of new treatments for any epidemic outbreak is of great interest and in vitro, biochemical assays are the basis of drug discovery. The recent outbreak of the coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands an affordable and reliable assay for testing antivirals. Here, we developed a quick and inexpensive high-throughput fluorescent assay to test inhibitors of viral proteases. Accordingly, we employed this assay to sample inhibitors for papain-like protease from SARS-CoV-2. In addition, we validated this assay for screening inhibitors of flaviviral protease from the tick-borne encephalitis virus to emphasize a broad range of applications of our approach. This fluorescent high-throughput assay is based on fluorescent energy transfer (FRET) between two distinct fluorescent proteins (eGFP and mCherry) connected via a substrate polypeptide. When the substrate is cleaved, FRET is abolished and the change in fluorescence corresponds to reaction progress. Our data show that this assay can be used for testing the inhibitors in the 96 or 384 well plates format with robust and reproducible outcomes. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.21.348821v1?rss=1 Authors: Murayama, A. Y., Kuwako, K.-i., Okahara, J., Bae, B.-I., Okuno, M., Mashiko, H., Shimogori, T., Walsh, C. A., Sasaki, E., Okano, H. Abstract: GPR56, a member of the adhesion G protein-coupled receptor family, is abundantly expressed in cells of the developing cerebral cortex, including neural progenitor cells and developing neurons. The human GPR56 gene has multiple presumptive promoters that drive the expression of the GPR56 protein in distinct patterns. Similar to coding mutations of the human GPR56 gene that may cause GPR56 dysfunction, a 15-bp homozygous deletion in the cis-regulatory element upstream of the noncoding exon 1 of GPR56 (e1m) leads to the cerebral cortex malformation and epilepsy. To clarify the expression profile of the e1m promoter-driven GPR56 in primate brain, we generated a transgenic marmoset line in which EGFP is expressed under the control of the human minimal e1m promoter. In contrast to the endogenous GPR56 protein, which is highly enriched in the ventricular zone of the cerebral cortex, EGFP is mostly expressed in developing neurons in the transgenic fetal brain. Furthermore, EGFP is predominantly expressed in GABAergic neurons, whereas the total GPR56 protein is evenly expressed in both GABAergic and glutamatergic neurons, suggesting the GABAergic neuron-preferential activity of the minimal e1m promoter. These results indicate a possible pathogenic role for GABAergic neuron in the cerebral cortex of patients with GPR56 mutations. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.08.329169v1?rss=1 Authors: Dong, A., He, K., Dudok, B., Farrell, J. S., Guan, W., Liput, D. J., Puhl, H. L., Cai, R., Duan, J., Albarran, E., Ding, J., Lovinger, D. M., Li, B., Soltesz, I., Li, Y. Abstract: Endocannabinoids (eCBs) are retrograde lipid neuromodulators involved in many physiologically important processes. However, their release and dynamics in the brain remain largely unknown, in part due to lack of probes capable of reporting real-time eCBs with sufficient spatiotemporal resolution. Here, we developed a new G protein-coupled receptor Activation Based eCB sensor GRABeCB2.0 using the human CB1 cannabinoid receptor and a circular-permutated EGFP. GRABeCB2.0 exhibited proper cell membrane trafficking, ~seconds kinetics, high specificity and robust fluorescence response to eCBs at physiological concentrations. Using GRABeCB2.0, we detected evoked eCB dynamics in both cultured neurons and acute brain slices. Interestingly, we also observed spontaneous compartmental eCB transients that spread ~11 m in cultured neurons, suggesting locally-restricted eCB signaling. By expressing GRABeCB2.0 in vivo, we readily observed foot-shock elicited and running triggered eCB transients in mouse amygdala and hippocampus, respectively. Lastly, using GRABeCB2.0 in an epilepsy model, we observed a spreading eCB wave following a calcium wave in mouse hippocampus. In summary, GRABeCB2.0 is a powerful new probe to resolve eCB release and dynamics under both physiological and pathological conditions. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.11.245753v1?rss=1 Authors: Gessara, I., Dittrich, F., Hertel, M., Hildebrand, S., Pfeifer, A., Frankl-Vilches, C., McGrew, M., Gahr, M. Abstract: SUMARYThe ability to genetically manipulate organisms has led to significant insights in functional genomics in many species. In birds, manipulation of the genome is hindered by the inaccessibility of the one-cell embryo. During embryonic development, avian primordial germ cells (PGCs) migrate through the blood stream and reach the gonadal anlage; where they develop into mature germ cells. Here, we explored the use of PGCs to produce transgenic offspring in the zebra finch, which is a major animal model for sexual brain differentiation, vocal learning and vocal communication. Zebra finch PGCs (zfPGCs) obtained from embryonic blood significantly proliferated when cultured in an optimized culture medium and conserved the expression of germ and stem cell markers. Transduction of cultured zfPGCs with lentiviral vectors was highly efficient leading to strong expression of the enhanced green fluorescent protein (eGFP). Transduced zfPGCs were injected into the host embryo and transgenic songbirds were successfully generated. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.21.214452v1?rss=1 Authors: Almeida, M. P., Welker, J. M., Ekker, S. C., Clark, K. J., Essner, J. J., McGrail, M. Abstract: The Cre/lox recombinase system has been widely used for spatiotemporal control of gene expression in animal model systems, however, efficient methods to isolate zebrafish Cre drivers that reliably recapitulate endogenous expression patterns of known genes are in need. Here, we apply CRISPR/Cas9 double-strand breaks to direct targeted integration using short homology of a 2A-Cre recombinase transgene into the early coding region of proneural genes ascl1b, olig2 and neurod1. The pPRISM-2A-Cre targeting vector contains a linked lens-specific fluorescent secondary marker cassette for identification of transgenic embryos, and we observed high rates of germline transmission ranging from 10%-100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The established transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase. in situ hybridization showed Cre expression reflected the pattern of the endogenous targeted proneural gene. In combination with Tg(ubi:loxP-EGFP-loxP-mCherry), each driver induced a switch from EGFP to mCherry expression in the expected proneural cell populations and their descendants, indicating efficient Cre-mediated recombination and excision of the floxed EGFP cassette. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre drivers whose spatial and temporal restricted expression mimics endogenous genes, surmounting the challenges associated with promoter BAC cloning and transposon mediated transgenesis. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.12.088450v1?rss=1 Authors: Ghezzi, F., Marques-Smith, A., Anastasiades, P. G., Lyngholm, D., Vagnoni, C., Rowett, A., Hoerder-Suabedissen, A., Nakagawa, Y., Molnar, Z., Butt, S. J. B. Abstract: Subplate neurons (SPNs) are a transient neuronal population shown to play a key role in nascent sensory processing relaying thalamic information to the developing cerebral cortex. However there is little understanding of how heterogeneity within this population relates to emergent function. To address this question we employed optical and electrophysiological technologies to investigate the synaptic connectivity of SPNs defined by expression of the Lpar1-EGFP transgene through the first postnatal week in primary whisker somatosensory cortex (S1BF). Our data identify that the Lpar1-EGFP SPNs represents two morphological subtypes: (1) transient, fusiform SPNs with axons largely restricted to the subplate zone; (2) pyramidal SPNs with axon collaterals that traverse the overlying cortex to extend through the marginal zone. Laser scanning photostimulation of caged glutamate was used to determine columnar glutamatergic and GABAergic input onto both of these SPN subtypes. These experiments revealed that the former receive translaminar input from more superficial cortical layers up until the emergence of the whisker barrels (~postnatal (P)5). In contrast, pyramidal SPNs only receive local input from the adjacent subplate network at early ages but then at later ages can acquire varied input from the overlying cortex. Combined electrical stimulation of the ventral posterior nucleus of the thalamus and optogenetic activation of thalamic afferents in thalamocortical slice preparations revealed that Lpar1-EGFP SPNs only receive sparse thalamic innervation during early postnatal development. Taken together, these data reveal two components of the postnatal network that interpret sparse thalamic input to direct the emergent columnar structure of neonatal somatosensory cortex. Copy rights belong to original authors. Visit the link for more info

For more information, please visit: http://bitesizebio.com/webinar/25961/deciphering-steps-of-mrnp-assembly-in-developing-oocytes-using-super-resolution-microscopy/ All mRNA molecules recruit specific proteins to form ribonucleoprotein complexes (mRNPs). Composition and localization of many mRNPs change dynamically from translation to decay. Microscopic techniques with high spatial and temporal resolution are invaluable for studying mRNP biogenesis. We have developed new tools based on fluorogenic forced intercalation (FIT) probes for RNA detection, quantification and interference in biological samples. The probes contain a thiazole orange (TO) dye introduced at a position normally occupied by a nucleobase. Upon binding to target nucleic acids, the TO dye increases its quantum yield and brightness substantially (greater than10 fold). These probes detect mRNA in a rapid, wash-free FISH setup using conventional wide-field microscopy. It is an ideal tool for RNA localization screens. Nuclease resistant FIT probes containing a locked nucleic acid adjacent to the TO dye are bright and contrasted enough for use in live imaging. These probes can also be designed to target functional elements of RNAs to test the role of those in RNP biogenesis. Absorption and emission spectra of TO are sufficiently different from EGFP to enable high sensitivity and specificity RNA-protein co-localization analysis, even with super-resolution, to study the RNA interactome. LNA modified FIT probes are excellent subjects for STED microscopy as duplex formation greatly increases their fluorescence lifetime.

The CHO-K1 cell line is the most common expression system for therapeutic proteins in the pharmaceutical industry. Due to the nature of economics, the cell lines and the vector design are subject to constant change to increase product quality and quantity. During the cultivation, the production cell lines are susceptible to decreasing productivity over time. Often the loss of production can be associated with a reduction of copy number and the silencing of transgenes. During cell line development, the most promising cell lines are cultivated in large batch culture. Consequently, the loss of a stable production cell line can be very cost-intensive. For this reason I developed different strategies to avoid a reduced productivity. Instability of production cell lines can be predicted by the degree of CpG methylation of the driving promoter. Considering that the DNA methylation is at the end of an epigenetic cascade and associated with the maintenance of the repressive state, I investigated the upstream signals of histone modifications with the assumption to obtain a higher predictive power of production instability. For this reason I performed a chromatin immunoprecipitation of the histone modifications H3K9me3 and H3K27me3 as repressive signals and H3ac as well as H3K4me3 as active marks. The accumulations of those signals were measured close to the hCMV-MIE at the beginning of the cultivation and were then compared with the loss of productivity over two month. I found that the degree of the H3 acetylation (H3ac) correlated best with the production stability. Furthermore I was able to identify an H3ac threshold to exclude most of the unstable producers. In the second project I aimed to improve the vector design by considering epigenetic mechanisms. To this end I designed on the one hand a target-oriented histone acetyltransferase to enforce an open and active chromatin status at the transgene. On the other hand I point-mutated methylation-susceptible CpGs within the hCMV-MIE to impede the maintenance of inactive heterochromatin formation. Remarkably, the C to G mutation located 179 bp upstream of transcription start site resulted in very stable antibody producing cell lines. In addition, the examination of cell pools expressing eGFP showed that G-179 promoter variants were less prone to a general methylation and gene amplification, which illustrates the dominating effect in epigenetic mechanisms of one single CpG. The last project was performed to localize stable integration sites within the CHO-K1 genome. In so doing I could show that the transfection leads predominantly to integration into inactive regions. Furthermore I identified promising integration sites with a high potential to induce stable expression. However, those results are preliminary and must be viewed with caution. Further examination needs to be done to confirm these results. Considering the results of all three projects, I propose that the interplay of metabolic burden and selection pressure at an early time point of cultivation plays an important role in cell line development. Small alterations of selection pressure can lead to a decisive change of cell properties. Therefore, stable cells are less susceptible than weak producers. The increase of selection pressure leads to compensatory effect by gene amplification in the instable cell lines. The resulting adjustment of productivity masks the truly stable cells, which precludes the selection of the right cell lines. For this reason the selection pressure, the copy number as well as the growth rate should be considered to minimize repressive effects.

Myelin Oligodendrocyte Glycoprotein (MOG) is one of the few proteins known to be localized on the outermost sheath of central nervous system (CNS) myelin. Due to this localization, MOG is accessible to antibodies. Anti-MOG antibodies are demyelinating and enhance clinical symptoms in a number of animal models of CNS inflammation. Autoantibodies recognizing conformationally intact MOG are found in different inflammatory diseases of the CNS, but their antigenic epitopes had not been mapped. In this work, 9 variants of MOG with an intracellular enhanced green fluorescent protein (EGFP) tag were expressed on the cell surface of human HeLa cells and used to analyze sera from 111 patients (104 children, 7 adults), who had antibodies recognizing cell-bound human MOG. These patients had different diseases, namely acute disseminated encephalomyelitis (ADEM), one episode of transverse myelitis or optic neuritis, multiple sclerosis (MS), anti-aquaporin-4 (AQP4)–negative neuromyelitis optica (NMO), and chronic relapsing inflammatory optic neuritis (CRION). The expression levels of the mutants were comparable and cells with a defined expression level (fluorescence intensity in the EGFP channel of 102-103) were gated. Each MOG-mutant was recognized by at least one MOG-specific mAb. This allowed the comparison of binding to the different mutants. In order to assess the reproducibility of the system, binding of the 111 sera to the mutants was analyzed up to three times in independent experiments, yielding a very good reproducibility of the binding percentage with an absolute SD of 7.8% in the case of low recognition of a mutant and a relative SD of 20% in the case of high recognition of a mutant. The applied variants of MOG gave insight into epitope recognition of 98 patients. All epitopes identified in this work were located at loops connecting the ß-strands of MOG. The immunodominant epitope of human anti-MOG antibodies was at the membrane-proximal CC’-loop containing aa42, which is not present in rodent MOG. This loop was recognized by about half of all patients. Overall, seven epitope patterns were distinguished, including the one mainly recognized by mouse mAbs at the FG-loop around aa104. Evidence from mouse models of CNS inflammation shows that anti-MOG antibodies recognizing different epitopes can be demyelinating and thus pathogenic. This suggests that not only those antibodies recognizing the same epitope of MOG as the pathogenic mAbs (i.e. the FG-loop), but also the ones recognizing the CC'-loop are pathogenic in humans, as both epitopes allow for the recognition of cell-bound MOG. In half of the patients, the anti-MOG response was directed to a single epitope. To analyze the effect of glycosylation on the recognition of MOG by human autoantibodies, a “non-glycosylation mutant” N31D was made. Digestion with PNGaseF and Western blot analysis confirmed that N31 was the only used N-glycosylation site of the MOG constructs in HeLa cells. Glycosylation of MOG was not needed for antibody binding, but 8% of the patients recognized deglycosylated MOG at least two-fold better. The epitope specificity was not linked to certain disease entities. The individual epitope recognition patterns stayed constant in 11 analyzed patients over an observation period of up to 5 years without evidence for intramolecular epitope spreading. Some patients with acute syndromes had anti-MOG IgG at disease onset, but rapidly lost their anti-MOG IgG reactivity. These patients were able to generate a long-lasting IgG response to measles and rubella virus vaccine indicating that the loss of anti-MOG reactivity was not reflective of a lack of capacity for longstanding IgG responses. Human anti-MOG antibodies are mainly of the IgG1 isotype, which can activate complement and antibody dependent cellular cytotoxicity. Upon binding to MOG in the CNS, human anti-MOG antibodies are hence expected to cause demyelination. Transfer experiments with purified human anti-MOG antibodies have not been performed yet. The fact that the majority of human anti-MOG antibodies did not recognize rodent MOG has implications for animal studies. Using the described assay will help to identify patient samples appropriate for these transfer experiments and finally lead to the formal proof of the pathogenicity of human anti-MOG antibodies. This work also gives important information for future detection of potential mimotopes and the development of anti-MOG antibody detection assays and might pave the way to antigen-specific depletion.

Confocal microscopy is known for its capability to produce exceptional 3D images, even in living tissue. At the same time, it is a powerful spectroscopic tool, facilitating fluores- cence methods such as Fluorescence Correlation Spectroscopy (FCS) or single-molecule Förster Resonance Energy Transfer (FRET). It is heavily used to investigate a wide range of biological problems. This holds true especially for protein properties such as ligand binding, complex formation, conformational changes, or the intracellular distribution of the species in question. In this work, I will describe the assembly of two instruments: The first is a multi- parameter fluorescence detection (MFD) setup. It is a purely spectroscopic tool that offers the capability to characterize a fluorescent molecule, delivering information like fluorescence lifetime, anisotropy or the speed of its diffusion in free solution. When the molecule of interest is labelled with two fluorophores, additional information, like the energy transfer in-between them, becomes accessible and the correct distance between these two fluorophores can be calculated. If the two fluorophores are attached to different molecules, the MFD setup can detect interactions of these molecules in the range from pM up to μM with the help of Fluorescence Cross-Correlation Spectroscopy (FCCS). The second instrument, a stimulated emission depletion setup, combines some of the mentioned techniques, like FCS, with the superior image capability of a confocal micro- scope. One particular problem of fluorescent microscopes, though, is that image resolution is always restricted to the diffraction limit of the wavelength of the laser light. The STED setup utilizes the effect of stimulated emission in order to circumvent the diffraction bar- rier and allows images with a three-fold resolution increase, down to 75nm. These two setups will be used for several applications: The first will be centered around the molecular conformation of proteins, which are sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. I will apply single-pair FRET to a small 29 amino acid long model peptide to investigate unfolding mechanisms of different unfolding reagents from the Hofmeister series, like sodium perchlorate or guanidinium chloride. The results show that certain salts, which are commonly summarized as denaturing agents, achieve the unfolding by either collapsing the molecule to a compressed state or swelling it to a denatured state. 7 The second application of the MFD setup is the investigation of the enhanced green fluorescent protein (EGFP). Although highly used in biochemistry and biophysics, for example to read out the expression level of genes, it is still not fully known what percentage of EGFP is fluorescent. This lack of knowledge makes it nearly impossible to make quantitative statements. With the help of FCCS, it is shown that the folding efficiencies range from 40 − 90%, depending on the environment of the fluorescent protein and which particular mutant is used. In the third application, the focus will be shifted to nucleation- and polymerization- behavior of actin. The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell di- vision. In order to compare results of confocal spectroscopy methods with well-established bulk essays, we successfully ported the standard bulk essay to the confocal microscope, allowing for the first time to follow the decrease of monomer concentration and appear- ance of small filaments. Also, the formation of dimers or other small oligomers below the critical concentration is proven for the first time, using FCCS. The last application will utilize the STED setup in order to carry out the first steps towards the investigation of the nucleation and branching behavior of actin in cooperation with the actin related protein 2/3 (ARP2/3). This protein complex preferentially attaches to actin filaments that are located at the leading edge of a cell and forms branched filamentous structures. The exact conditions under which this process occurs are not well characterized. This part of the work will deal with the steps that are necessary to follow the polymerization process on the STED setup.

The ability to adapt to environmental changes, to learn and to memorize information is one of the brain’s most extraordinary features. One important process underlying this ability is considered to be synaptic plasticity, i.e. the structural and functional modification of synaptic connections. Synaptic plasticity can occur either by genesis or elimination of synaptic connections, or at existing connections by modifications in the strength of synaptic transmission. Synaptic connections are complex entities consisting of different functional structures: The majority of hippocampal and cortical excitatory synapses are made up of a postsynaptic compartment called dendritic spine and a presynaptic compartment called bouton. Within the spine and the bouton dense molecular structures, which serve the synaptic transmission between pre‐ and postsynapse, exist, namely the postsynaptic density (PSD) in the spine, and the active zone (AZ) in the bouton. All these structures are correlated in size and with synaptic strength. The function of this correlation serves the efficient and fast transmission of neuronal signals. During synaptic plasticity, a coordinated change in the size of all synaptic structures is expected, for the maintenance of their correlation. However, to date, such coordinated modifications have not been examined in detail. Furthermore, the mechanisms underlying the maintenance of structural and functional changes after synaptic plasticity remain poorly understood. The aim of this thesis was to explore these questions. To achieve this I carried out two complementing experimental approaches: In a first set of experiments, I studied changes in spine and PSD size by twophoton time‐lapse imaging to explore correlated modifications in these two synaptic structures. To induce structural spine plasticity I stimulated single dendritic spines of Schaffer collateral synapses in cultured hippocampal slices by two‐photon glutamate uncaging. This was shown previously to be accompanied by an increase in spine size and synaptic strength. To visualize structural plasticity of spines and their PSD, the cytosolic marker tdTomato and EGFP‐tagged structural proteins of the PSD, namely PSD‐95 and Homer1c, were co‐expressed. PSD‐95 and Homer1c are important and abundant scaffolding proteins of the PSD, which have been used previously as markers for PSD size. I found that both PSD‐95 and Homer1c levels increased after spine stimulation. Homer1c increased rather rapidly whereas PSD‐95 did so in a delayed manner relative to the increase in spine volume. Thus, the naïve correlation between PSD protein level and spine volume was only transiently disrupted after plasticity induction, but was reestablished over a time course of 3 hours. Furthermore, PSD‐95 level only increased significantly in spines with persistent enlargement, but not in spines with non‐persistent enlargement. On the other hand, Homer1c level initially increased both in spines with and without persistent enlargement, and then decayed back to original level in spines with non‐persistent enlargement. Because the increase in PSD‐95 level was delayed, I investigated whether the application of the PKA activator forskolin, which supports an increased and persistent enlargement of spines after glutamate uncaging, might promote and therefore accelerate an increase in PSD‐95 level. However, these experiments led to unexpected results: forskolin application neither had an effect on spine volume nor on PSD‐95 level increase. Although PSD‐95 and Homer1c are important and abundant PSD scaffolding proteins, they represent only two out of a multitude of proteins which form the PSD. Consequently, an increase in the PSD marker proteins does not necessarily represent an increase of the PSD as a whole. Therefore, in a second experimental approach, I applied electron microscopy to stimulated spines which displayed a stable enlargement over 3 hours after stimulation. Hereby, I was able not only to reconstruct the spine and the entire PSD, but also the bouton at the stimulated spine: I found that spine, PSD and bouton displayed matching dimensions 3 hours after stimulation, similar to naïve, unstimulated spines. In summary, by combining two‐photon glutamate uncaging with time‐lapse imaging and electron microscopy, I found that spine, the PSD and bouton increase during structural plasticity, and that the correlation between these structures is reestablished after stimulation on a time scale of 3 hours. Furthermore, an increase of synaptic structures correlates with the stabilization of synaptic modifications after plasticity. This suggests a model where the balancing of synaptic structures is a hallmark for the stabilization of structural modifications during synaptic plasticity.

Chronische Extremitätenischämien stellen eine sowohl subjektiv belastende als auch volkswirtschaftlich bedeutende Krankheitsentität dar. Dabei können etliche Patienten mit den zur Verfügung stehenden konventionellen Verfahren nicht befriedigend therapiert werden. Neuere Konzepte zur Zelltherapie der chronischen Therapie führten in der klinischen Erprobung dabei zu eher ausbaufähigen Resultaten. Unsere Gruppe konnte in Vorarbeiten zeigen, dass die exogene Applikation embryonaler Endothelprogenitorzellen (eEPCs) im Tiermodell zu einem deutlichen Effet auf die chronische Ischämie führt. Um diesen Effekt weiter zu steigern, wurde ein künstliches Fusionsmolekül aus dem Chemokin SDF-1 als Kopf, der Mucindomäne des Fractalkine als Rückgrat und einem GPI-Teil zur Verankerung im Endothel (SDF-Fractalkine-GPI oder S1FG) kloniert. Wir konnten ebenfalls in Vorarbeiten zeigen, dass dieses S1FG eEPCs in vitro und in vivo rekrutiert, und dass eine Vortransfektion des Endothels des Ischämiegebietes vor Applikation der eEPCs zu einer Steigerung des funktionellen Effekts führt. Wir stellten die Hypothese auf, dass ein Ersatz der exogenen Applikation der eEPCs durch eine Mobilisierung endogener vaskulärer Progenitorzellen ebenfalls zu einem guten funktionellen Effekt führt. Weiterhin sollte der genaue Rekrutierungsmechanismus des S1FG untersucht werden. Zur Untersuchung des funktionellen Effekts wurde wie in den Vorarbeiten ein Kaninchenmodell der chronischen Hinterlaufischämie gewählt, bei dem an Tag 0 die rechte Femoralarterie entfernt wurde. Nach Entwicklung eines chronischen Zustandes wurde am Tag 7 eine Angiographie beider Femoralisstromgebiete durchgeführt und entweder S1FG oder eGFP liposomal per Retroinfusion transfiziert. An den Tagen 9, 10 und 11 wurde jeweils 1 mg des kurzwirksamen CXCR4-Antagonisten AMD3100 oder 1 ml NaCl intraperitoneal injiziert. An Tag 35 wurde eine erneute Angiographie durchgeführt, das Tier getötet und die Hinterlaufmuskulatur entnommen. Die Angiogenese wurde über die mittels PECAM-1-Färbung bestimmte Kapillardichte gemessen, die Arteriogenese über die Mengenzunahme der in der Angiografie sichtbaren Kollateralen. Zur Messung der Perfusion wurden die Flussgeschwindigkeit in der Angiographie sowie fluoreszierende Mikrosphären verwendet. Um das Rekrutierungsprofil des S1FG in vitro zu eruieren, wurden statische Adhäsionsversuche mit PMNs auf transfizierten HMECs sowie Adhäsionsversuche in Flusskammern von THP-1-Zellen auf transfizierten HUVECs verwendet. Es zeigte sich, dass signifikant weniger PMNs auf S1FG-transfiziertem Endothel adhärieren, als auf Fractalkine-transfizierten HMECs, während die eEPC-Adhäsion signifikant besser war. Bei den Versuchen unter Schubspannung ergab sich durch S1FG-Transfektion keine signifikante Änderung der Anzahl rollender Zellen, während die Anzahl fest haftender Zellen signifikant höher war. Durch Zugabe eines L-Selektin-Antikörpers zu den THP-1-Zellen vor Superfusion konnte die Anzahl fest haftender Zellen wieder auf Kontrollniveau reduziert werden, während die Anzahl rollender Zellen leicht reduziert wurde. Zugabe von AMD3100 führte dort nicht zu einer signifikanten Änderung der Anzahl adhärierender Zellen, jedoch wurde durch AMD3100 die Stärke der Interaktion, gemessen als Anzahl nach Applikation hoher Flüsse noch adhärierender Zellen, wieder auf Kontrollniveau reduziert. Ein über andere Adhäsionsmoleüle vermitteltes Zellrollen ist also vermutlich eine Voraussetzung für eine adäquate S1FG-Funktion. Dabei würde es über SDF-1-CXCR4-Interaktion zu einer Erhöhung der Festigkeit der Bindung kommen. In Bezug auf den funktionellen Effekt im Tiermodell führte Verwendung von S1FG und AMD3100 zu einer signifikanten Steigerung von Kapillardichte, Kollateralenwachstum und Perfusion gegenüber der Kontrolle. Die Werte lagen dabei im selben Bereich wie durch Verwendung von S1FG und eEPCs erzielte Ergebnisse. Transfektion von S1FG ohne weitere Behandlung führte nicht zu einer signifikanten Änderung eines Parameters zur Kontrolle, während die Verwendung von AMD3100 zu moderaten Steigerungen bei Kapillardichte, und Kollateralenwachstum führte. Die Werte waren jedoch immer noch signifikant geringer als die nach Kombination von S1FG und AMD3100 erreichten Werte. Verwendung einer proteaseresistenten, funktionell jedoch aktiven Mutante für das SDF-1 im S1FG-Molekül führte nicht zu signifikanten Änderungen bei funktionellen Parametern, bis auf eine zwar signifikante, quantitativ jedoch geringe Verringerung des Kollateralwachstums. Zusammenfassend kann man sagen, dass die lokale Applikation eines künstlichen Adhäsionsmoleküls gemeinsam mit einer Mobilisierung knochenmarksständiger endothelialer oder vaskulärer Progenitorzellen zu einem deutlichen funktionellen Effekt führt, der den der Zellmobilisierung ohne Adhäsionssteigerung übertrifft. Dennoch birgt der Ansatz einige Risiken, wie die versehentliche Förderung von Tumorangiogenese oder die Beschleunigung des Wachstums atherosklerotischer Plaques. Zusätzlich bleibt unklar, welche Zellen genau durch das Adhäsionsmolekül rekrutiert werden, und ob es sich überhaupt um eine homogene Zellpopulation handelt. Weiterhin bleibt zu überprüfen, in welcher Weise die Wirksamkeit der Therapie durch chronische Defekte der Progenitorzellmobilisierung und -funktion, wie sie beispielsweise bei Diabetes oder Nikotinabusus auftreten, beeinträchtigt wird, und ob gegebenenfalls Optimierungsmöglichkeiten in Bezug auf das Mobilisierungsregime, den Aufbau des Adhäsionsmoleküls oder die Applikationsart bestehen. Nichtsdestotrotz stellt diese Methode einen vielversprechenden neuen Ansatz zur Verbesserung der bisher eher zwiespältigen Ergebnisse der Zelltherapie dar.

Auditory stimuli are processed by several parallel and serial neural circuits in the auditory brainstem. In the first part of this PhD thesis, synaptic integration of excitatory inputs in the neural network of the dorsal nucleus of the lateral lemniscus (DNLL) in Mongolian gerbils is investigated. The second part of this study analyses the feasibility of the use of viral vectors in Mongolian gerbils. This work aims to add to the available methods for neural circuit analysis in these animals by establishing tools for genetic manipulation. The DNLL receives excitatory inputs from the superior olivary complex (SOC) and provides GABAergic inhibition to its contralateral counterpart and both inferior colliculi (ICs). This GABAergic inhibition can outlast the triggering auditory stimulus by tens of milliseconds and thus differs substantially from the fast glycinergic inhibition prevailing in the SOC. It is thought that this persistent inhibition (PI) suppresses further processing of sound source information cues of lagging sounds, thereby providing the neuronal basis for sound localisation in reverberant environments. The mechanisms which PI is generated are still under debate. One hypothesized mechanism focuses on the output mechanism in DNLL neurons, favouring transmitter spillover or asynchronous release to evoke PI. A second mechanism states that integration of excitatory inputs leads to temporally extended activity in DNLL neurons, thereby prolonging the GABAergic output. Here, we tested in vitro the feasibility of the integration based mechanism in Mongolian gerbils. We analyzed the integration of excitatory inputs to DNLL neurons and found that five simultaneously stimulated excitatory fibres, each releasing on average ~18 vesicles are sufficient to trigger a single action potential (AP) in a DNLL neuron. A strong presynaptic stimulation pulse could trigger multiple APs. The input-output functions (IO-Fs) of DNLL neurons were dependent on NMDA receptor (NMDAR) mediated currents, which temporally extended the neuron's activity. The synaptic IO-Fs of DNLL neurons could also be modulated by voltage gated potassium, but not by calcium conductances.The NMDAR dependent activity amplification, which is maintained into adult stages, is shown to prolong the GABAergic output of DNLL neurons, thus contributing to PI generation. Viral vectors are widely used to alter the genetic content of a host organism. In Mongolian gerbils this approach may be suitable to compensate for the lack of genetic strategies in neural circuit analysis such as transgenic animal lines. Lentiviral and Semliki forest viral vectors were stereotactically injected into the IC or the medial nucleus of the trapezoid body (MNTB) in Mongolian gerbils. The lentiviral constructs were able to induce expression of the transgenic protein in the IC but not in MNTB principal neurons. The Semliki forest viral vector induced expression in both nuclei but also caused strong cytotoxic effects in the infected cells. In a further experiment, an eGFP expressing pseudorabies virus based on the attenuated Bartha strain (PRV-152) was stereotactically injected into the IC and was able to retrogradely infect the nuclei of the auditory brain stem in juvenile and adult Mongolian gerbils. PRV-152 spread synaptically to 2nd order neurons by ~20 hours after injection. Infection could also be started in the DNLL and showed a strongly pronounced neurotropism. The virus induced eGFP expression was high and allowed for a detailed visualization of the infected neurons, establishing PRV-152 as an effective tool for anatomical circuit analysis. The feasibility of using this virus in conjunction with electrophysiological investigations was also tested. 37% of 1st and 78% of 2nd order infected neurons show a significant decrease of excitability, which impedes the use of PRV-152 in combination with electrophysiological recordings for physiological analysis of neural circuits.

Intracellular protein delivery is offering numerous possibilities in research and in therapy. Aside gene therapy, protein delivery into living cells is one of the most promising tools for the treatment of various so far immedicable diseases including cancer. To develop a practicable protein delivery platform, a test system which allows easy control of successful intracellular delivery is needed. Therefore a test system based on two model proteins was established. A nuclear localization signal tagged EGFP molecule is enabling fast control of cellular uptake and endosomal release. The second model protein ß-galactosidase is evidencing that protein conformation is not irreversible disturbed by modification with the carrier molecules. Protein transduction technology is opening the door for a promising alternative to gene therapy, as it is lacking of the potential malignant side effects of gene therapy. The most limiting step in the development of a therapeutic drug remains the delivery process. In the last decade, many techniques to deliver proteins into living cells were developed. Although great efforts were made, so far no all-purpose technique is available that addresses all critical steps, like efficient uptake, endo-lysosomal escape, low toxicity, while maintaining enzymatic activity. Each method has got its limitation, for example cell type dependence. Among the so far used carriers, the most effective ones are cationic polymers like polyethylenimine. These carriers are lacking of precise structure and often show high toxicity, dependent on the molecular weight of the used polymer. In this thesis the properties of the three arm cationic oligomer 386, which was previously designed for siRNA delivery was investigated in regard of being applicable as a transduction carrier for protein delivery. This carrier molecule, in contrast to other cationic polymers used for protein delivery, is of precise structure, of low molecular weight and potentially degradable by proteases. The transduction oligomer was covalently bound to the protein by a bioreversible bond. Our results reveal that covalent coupling of the structure defined cationic oligomer 386 to a protein leads to a high efficient, serum insensitive and low toxic alternative to established protein transduction technologies. For a general all-purpose delivery system covalent coupling of the carrier to the cargo protein is indispensable. Protein delivery requires special properties to the linker molecule. Therefore in this work a new pH sensitive linker was developed which combines the advantages of click reactions with the implementation of a traceless cleavable bond between two conjugated molecules. Three different click chemistries were performed which all are compatible with the acid labile properties. A traceless cleavage may be a particularly important feature in protein transduction strategies, to maintain full bioactivity of enzymes and other proteins. The current example of 386 carrier-mediated cytosolic delivery and subsequent nuclear import of released nls-EGFP demonstrates the advantage of the traceless linker. To demonstrate that the modification does not irreversibly affect structure and biological activity of proteins, 386-AzMMMan-ßgalactosidase was delivered as a model enzyme. It exhibited cytosolic activity in the transduced cells far higher than without shuttle. Aside from these encouraging options for protein delivery and modification, the linker might have broader use in the design of novel programmed, acid labile and biodegradable drug delivery systems. Targeted therapeutics could, after delivery into acidic tumor areas or upon cellular uptake into endosomes, be dismantled from their outer shell including targeting ligands. Besides drug delivery, the linker may also be of interest for other applications, such as reversible labeling of various biological and also chemical molecules. The developed linking strategy and the presented concepts for transduction shuttles may help to get a step closer in the design of an all-purpose protein delivery platform, applicable on bench as on bedside.

Plastid transformation is a valuable technique for both basic and applied science. In basic science the technique is used to study chloroplast function. Applied approaches deal with the potential of the plastid for the production of medicinal therapeutics, in most cases vaccine antigens coupled to adjuvants. Adjuvants are used for the trans-mucosal delivery of attached cargoes. Cell penetrating peptides (CPPs) emerged as valuable tools for the delivery of cell-impermeable cargoes across cell barriers more than twenty years ago. Although the exact mechanism of CPP penetration of cells is still discussed, the applied value of CPPs is documented in a number of clinical studies. Recently, scientists working in the CPP field launched a call for an alternative expression platform for CPP fusion peptides / proteins. Only a short time before, CPPs were introduced into plant science and some impressive first results, manipulating plant cells from the “outside”, were achieved. The present study aimed at combining the fields of plastid transformation and CPPs from the “inside”. We report the first expression of CPP fusion proteins in a plant, more precisely in the plastid. The approach focused on three aspects of CPP fusion protein expression in the organelle: (A) the principal feasibility of CPP-fusion protein expression in the plastid, (B) the location of CPP fusion proteins in the plant cell upon plastid-based expression and (C) the use of plastids for the manufacture of CPP fusions to provide an alternative to the bacterial expression system. Nine prominent CPPs were employed in three vector series to investigate these aspects. In vector series I the selected CPPs were fused to the fluorescent protein eGFP to provide an optical read-out; in vector series II, the CPPs were fused to Arabidopsis MYB transcription factor PAP1 to provide a biological read-out and in vector series III, two CPPs were fused to the human enzyme PAH to introduce plant-based CPP fusion protein expression. Taken together, the expression of CPP fusion in the plastid turned out to be feasible. Transplastomic plants reached homoplasmy, produced viable seeds and stably inherited the desired trait to their progenies in a maternal fashion. Only low protein accumulation levels were detected. Pleiotropic effects occured at the low protein accumulation levels observed. Localisation of CPP fusion proteins was shown to be restricted to the plastid. An inability of CPP fusion proteins isolated from vector series I to penetrate protoplasts, young plant tissue and human cell lines was revealed. The value of a plastid-based manufacture of CPP fusion proteins for clinical approaches failed to be demonstrated due to low fusion protein accumulation levels. Bottlenecks of the current study are discussed and suggestions are made to provide a framework for future efforts.

Background: It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs. Results: N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Monitoring the fraction of viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes measured. Conclusions: The characterized double-labeled FVs described here provide new mechanistic insights into FV early entry steps, demonstrating that productive viral fusion occurs early after target cell attachment and uptake. The analysis highlights apparent differences in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled FVs promise to provide important tools for future detailed analyses on individual FV fusion events in real time using advanced imaging techniques.

Im Rahmen dieser Arbeit konnte gezeigt werden, dass der Notch-Signalweg auch in Hydra für die Ausbildung und Aufrechterhaltung zweier entwicklungsrelevanter Grenzen benötigt wird. Sowohl während der späten Knospung als auch während der Musterbildung eines Kopfes ist seine Aktivität für die Etablierung zweier benachbarter, scharf zueinander abgegrenzter Signalzentren verantwortlich. Es konnte gezeigt werden, dass dies Voraussetzung für die normale Entwicklung von Knospen aber auch für die Strukturierung eines Kopfes in seine drei Teilbereiche ist. Im Fall der Knospung war nach Inhibition des Notch-Signalweges die Knospenfußbildung inhibiert, weshalb sich die Knospen nicht an ihrer Basis einschnüren und vom Elterntier abspalten konnten. Dies resultierte in der Entwicklung stabiler Y-Tiere. Die Expression von HyHes und der Matrix-Metalloprotease MMP-A3 konnte in DAPT-behandelten Tieren nicht mehr detektiert werden. Dadurch konnte gezeigt werden, dass HyHes ein primäres Zielgen des Notch-Signalweges darstellt. Dies konnte auch in einem in vitro-Reportergenversuch bestätigt werden, indem HvNICD die Expression von EGFP ausgehend vom HyHes-Promotor über zwei Su(H)-Bindestellen induzieren konnte. Die Expression des FGF-Rezeptors kringelchen und des Gerüstproteins Hydsh war nach DAPT-Behandlung nicht inhibiert, sondern diffus und unbegrenzt. Eine Expression in klaren, streifenförmigen Domänen an der Knospenbasis wie in unbehandelten Tieren konnte nie beobachtet werden. Demnach wird die Aktivität des Notch-Signalweges im Verlauf der Knospung vermutlich für die Schärfung einer bestehenden Grenze zwischen Muttertier und Knospe benötigt. Dies ermöglicht die definierte, voneinander abgegrenzte Expression von Genen, die einerseits für die voranschreitende Einschnürung der Knospenbasis und andererseits für die Fußdifferenzierung der Knospe verantwortlich sind. Neben der Knospung wird der Notch-Signalweg auch für den Erhalt adulter Kopfstrukturen und für die de novo-Musterbildung eines Kopfes während der Kopfregeneration benötigt. Hvnotch wird in adulten an der Tentakelbasis (an der Grenze zwischen Tentakeln und Tentakelzone) und auch während der Kopfregeneration erhöht exprimiert. Die Inhibition des Notch-Signalweges führte auf morphologischer Ebene zu abnormen Kopfstrukturen und übermäßiger Produktion von Tentakelgewebe. Daraus konnte gefolgert werden, dass der Notch-Signalweg in unbehandelten Tieren die Ausbildung von Tentakelgewebe unterdrückt bzw. verhindert. Dies konnte auch auf molekularer Ebene durch Untersuchung der Expression von Hywnt3, HMMP und Hyalx gezeigt werden. Diese werden in unbehandelten Tieren je spezifisch in einem Teilbereich des Kopfes, nämlich dem Hypostom, den Tentakeln oder der Grenzregion zwischen Tentakeln und Tentakelzone exprimiert. In DAPT-behandelten adulten Tieren und Kopfregeneraten war ihre Expression inhibiert oder verändert und spiegelte die beobachteten Kopf-Fehlbildungen wider. Anhand von Transplantationsversuchen und der Untersuchung der Expression von Hywnt3 konnte überdies ein Einfluss des Notch-Signalweges auf die de novo-Ausbildung eines Kopforganisators während der Regeneration beobachtet werden.

Speaker: Dr. J. Petravic Abstract: In HIV dynamics models, it is commonly assumed that HIV-infected cells all have the same viral production and death rates. We explored the dynamics of viral production and death in vitro to determine the validity of this assumption. We infected human cells with HIV-1 constructs that expressed enhanced green fluorescent protein (EGFP) and determined the amount of viral proteins produced by infected cells. Analysis of the flow cytometry data showed that the productively infected cells exhibited a broad, approximately log-normal distribution of viral protein content (spanning several orders of magnitude) that changed its shape and mean fluorescence intensity over time, and that population death rate apparently did not correlate with its mean EGFP content. We assumed that the observed EGFP fluorescence level represented the balance of protein production and degradation. In our model of the infected cell population, EGFP fluorescence distribution at any time depended on probability distributions of four independent parameters: time to the start of protein production, protein production and degradation rates, and the lifespan of infected cells. After exploration of possible combinations of parameter distributions, we found that a distribution of protein production rates that is negatively correlated to the times to start of production of viral can explain the observed time course of the distribution of EGFP intensity.

Speaker: Dr. J. Petravic Abstract: In HIV dynamics models, it is commonly assumed that HIV-infected cells all have the same viral production and death rates. We explored the dynamics of viral production and death in vitro to determine the validity of this assumption. We infected human cells with HIV-1 constructs that expressed enhanced green fluorescent protein (EGFP) and determined the amount of viral proteins produced by infected cells. Analysis of the flow cytometry data showed that the productively infected cells exhibited a broad, approximately log-normal distribution of viral protein content (spanning several orders of magnitude) that changed its shape and mean fluorescence intensity over time, and that population death rate apparently did not correlate with its mean EGFP content. We assumed that the observed EGFP fluorescence level represented the balance of protein production and degradation. In our model of the infected cell population, EGFP fluorescence distribution at any time depended on probability distributions of four independent parameters: time to the start of protein production, protein production and degradation rates, and the lifespan of infected cells. After exploration of possible combinations of parameter distributions, we found that a distribution of protein production rates that is negatively correlated to the times to start of production of viral can explain the observed time course of the distribution of EGFP intensity.

Research applications and cell therapies involving genetically modified cells require reliable, standardized and cost-effective methods for cell manipulation. The goal of this work is to provide a novel methodology that produces, in a single standardized techonology, genetic modification and cell isolation. We have named this novel procedure ―Magselectofection”. The approach is based on magnetic cell separation and magnetically-guided gene delivery (magnetofection). Optimized gene vectors associated with novel magnetic nanoparticles were formulated to transfect/transduce target cells while they are passaged and separated through a high gradient magnetic field cell separation column. Magnetofection of the Jurkat T cells using selected vector formulations resulted in a significant (up to 4.5-fold) enhancement in both luciferase reporter gene expression and the percentage of cells expressing eGFP, as compared to lipofection. A procedure for vector loading on LS Miltenyi columns was developed that enables up to 100% retention for both non-viral and viral magnetic complexes. We demonstrate, using a model cell mixture of K562 and Jurkat T cells, that the integrated method is highly efficient and specific for the target cell population. This was not only true for the model Jurkat/K562 mixture, but also for Sca-1+ mouse hematopoietic stem cells. With human umbilical cord mesenchymal stem cells (hUC-MSCs), we achieve up to 30% transfected cells with non-viral vector doses as low as 8 pg plasmid DNA per cell and up to 100% transduced cells with a multiplicity of infection of 0.5 TU/cell using lentivirus. Similarly, we obtain 22% eGFP-positive human cord blood hematopoietic stem cells (hCB-HSCs) upon lentiviral magselectofection compared to 0.15% eGFP-positive cells post-standard infection. We achieve up to 50% transduced Sca-1+ mouse stem cells at a lentiviral MOI of 1-3. Up to 5-15% and 20% genetic modified PBMC were found using non-viral and viral magselectofection, respectively. After genetic modification using magselectofection differentiation potential of hCB-HSCs and hUC-MSCs was maintained. Magselectofection requires a minimal number of manipulation steps and results in efficient and specific gene delivery to target cells. This minimizes the necessary vector material while maintaining the cellular differentiation potential of modified stem cells. Magselectofection may become a useful tool for nucleic acid therapy approaches involving ex-vivo genetically modified cells.

microRNAs (miRNAs) are small non-coding RNAs of 21-24 nt in size, which are endogenously expressed in higher eukaryotes and play important roles in processes such as tissue development and stress response and in several diseases including cancers. In mammals, miRNAs guide proteins of the Argonaute family (Ago proteins) to partially complementary sequences typically located in the 3’-untranslated regions (3’-UTRs) of specific target mRNAs, leading to translational repression or mRNA degradation. To gain further insight into the function of human miRNAs, we analyzed the protein as well as the RNA composition of miRNA-Ago protein complexes in molecular detail. To identify novel Ago-interacting proteins, we isolated Ago complexes and investigated them by mass spectrometry and co-immunoprecipitation experiments. We found that trinucleotide repeat-containing 6B (TNRC6B), Moloney leukemia virus 10 (MOV10), RNA binding motif protein 4 (RBM4) and Importin 8 (Imp8) interact with human Ago proteins. Moreover, using RNA interference and EGFP and dual luciferase reporter assays, we demonstrated that these factors are required for miRNA function, indicating that we have identified new components of the miRNA pathway. Intriguingly, depletion of Imp8 does not affect the levels of mature miRNAs or the interaction of miRNAs with Ago proteins, but is required for efficient association of Ago-miRNA complexes with their target mRNAs. Thus, Imp8 is the first factor acting at the level of target mRNA binding, establishing a novel layer of regulation for the miRNA pathway. Imp8 is an Importin-β-like protein, which has previously been implicated in nuclear import of substrate proteins. In line with these results, we demonstrated that a detectable fraction of Ago2 localizes to the nucleus of human cells. Moreover, knockdown of Imp8 by RNAi reduces the nuclear signal of Ago2, suggesting that Imp8 affects the nuclear localization of Ago2. Therefore, our data suggest that Imp8 has a dual function both in the cytoplasmic miRNA pathway and in nuclear transport of Ago proteins. To identify small RNAs, which associate with human Ago proteins, we isolated, cloned and sequenced small RNAs bound to Ago1 and Ago2 complexes. In addition to known miRNAs, we found several small RNAs, which derive from small nucleolar RNAs (snoRNAs). We therefore investigated the function of one particular small RNA, which is derived from the snoRNA ACA45 and showed that it functions like a miRNA. Interestingly, this small RNA is processed by the miRNA maturation factor Dicer, but does not require the microprocessor complex that is essential for processing of primary miRNA transcripts. Thus, we have identified a novel biogenesis pathway of a new class of small RNAs that can function like miRNAs. To experimentally identify mRNAs that are stably associated with miRNA-Ago protein complexes, we isolated and analyzed Ago1 and Ago2-bound mRNAs by cloning and sequencing and by microarray hybridization techniques. Using dual luciferase reporter assays, we demonstrated that many Ago-associated mRNAs are indeed miRNA targets. Therefore, we have developed a method allowing for the identification of miRNA target mRNAs from cell lines or tissues of interest independently of computational predictions. In a project that was independent of our studies on Ago protein complexes, we investigated structural and functional requirements for the activity of small interfering RNAs (siRNAs). siRNAs are small double-stranded RNAs of appr. 21 nt in size, which trigger the sequence-specific endonucleolytic degradation of perfectly complementary target transcripts upon binding to Ago2. However, both single strands of a siRNA duplex can potentially have unwanted “off-target effects” by repressing partially complementary target mRNAs through binding to their 3’-UTRs. We therefore developed a method to selectively inhibit the activity of the siRNA strand that is dispensable for target silencing (“passenger strand”) through chemical modification of its 5’-end. This method could be a useful tool for the design of highly specific siRNAs. Taken together, we have analyzed the composition of Ago-miRNA protein complexes by a variety of methods and identified novel protein factors of the miRNA pathway, a novel class of small RNAs as well as a panel of previously unknown miRNA target mRNAs. The techniques for the purification and the analysis of Ago complexes that were developed in this study will provide useful tools for future analyses of miRNA pathway factors, small RNAs and miRNA target mRNAs from any tissue or cell line of interest.

Background: Pancreatic infiltration by leucocytes represents a hallmark in acute pancreatitis. Although leucocytes play an active role in the pathophysiology of this disease, the relation between leucocyte activation, microvascular injury and haemorrhage has not been adequately addressed.Methods: We investigated intrapancreatic leucocyte migration, leucocyte extravasation and pancreatic microperfusion in different models of oedematous and necrotising acute pancreatitis in lys-EGFP-ki mice using fluorescent imaging and time-lapse intravital microscopy.Results: In contrast to the current paradigm of leucocyte recruitment, the initial event of leucocyte activation in acute pancreatitis was represented through a dose- and time-dependent occlusion of pancreatic capillaries by intraluminally migrating leucocytes. Intracapillary leucocyte accumulation (ILA) resulted in dense filling of almost all capillaries close to the area of inflammation and preceded transvenular leucocyte extravasation. ILA was also initiated by isolated exposure of the pancreas to interleukin 8 or fMLP, demonstrating the causal role of chemotactic stimuli in the induction of ILA. The onset of intracapillary leucocyte accumulation was strongly inhibited in LFA-1-/- and ICAM-1-/- mice, but not in Mac-1-/- mice. Moreover, prevention of intracapillary leucocyte accumulation led to the development of massive capillary haemorrhages and transformed mild pancreatitis into lethal haemorrhagic disease.Conclusions: ILA represents a novel protective and potentially lifesaving mechanism of haemostasis in acute pancreatitis. This process depends on expression of LFA-1 and ICAM-1 and precedes the classical steps of the leucocyte recruitment cascade.

Das Tollwutvirus (Rabies Virus (RV)) ist ein neurotropes Virus aus der Familie der Rhabdoviridae innerhalb der Ordnung Mononegavirales. RV Partikel werden an der Zelloberfläche durch Membran-Budding gebildet. Dabei wird der virale Ribonukleoproteinkomplex (RNP) durch das Matrixprotein (M) in eine Membranhülle verpackt in der Trimere des Transmembran-Glykoproteins (G) selektiv inkorporiert werden. Die G Spikes vermitteln die Bindung der Virionen an zelluläre Rezeptoren und die Fusion der viralen und zellulären Membran. Auch in Abwesenheit des G werden Viren freigesetzt, da der Budding-Prozess hauptsächlich durch das M Protein vermittelt wird. Das G-unabhängige Budding der Rhabdoviren erlaubt den Austausch der Oberflächenproteine (Envelope-Switching), wodurch es zu einem veränderten Tropismus (Re-targeting) der RV kommt. In dieser Arbeit konnte gezeigt werden, dass die G Proteine der Lyssaviren Mokola, Bat Hamburg und Lagos Bat, aber auch des RV-Stammes Challenge Virus Strain (CVS) für den spezifischen Neurotropismus dieser Viren verantwortlich sind. Der für die Inkorporation von Fremdproteinen in die Virushülle essentielle Bereich des RV G (tm) wurde verwendet, um chimäre Typ I Transmembranproteine aus dem rot-fluoreszierenden Protein (RFP) und RV G (tm-RFP) zu konstruieren und in die Virushülle G-defizienter und Wildtyp Tollwutviren zu inkorporieren. Fusionsproteine aus tm und zellulärem Prionprotein (tm-PrPC) wurden nicht an die Bereiche der Zellmembran transportiert, an denen Tollwutvirus-Budding stattfindet. Diese Konstrukte könnten daher verwendet werden, um den Ort des RV-Budding näher zu bestimmen, während die Inkorporation fluoreszierender Proteine in die Virushülle die direkte Visualisierung von einzelnen Stadien des Tollwutvirus-Lebenszyklus wie z.B. Virustransport oder Entry in lebenden Zellen ermöglicht. Aufgrund der G-vermittelten transsynaptischen Ausbreitung der Rhabdoviren in neuronalen Netzwerken, wurden die Fluoreszenz-markierten RV Partikel auch als neuronale Marker zur Untersuchung der Physiologie und Morphologie neuronaler Netzwerke verwendet. Der retrograde, axonale Transport der RV zum Zentralen Nervensystem ist eine essentielle Voraussetzung für die letale RV-Erkrankung. Durch die Verwendung von RV Partikeln mit tm-RFP-markierter Virushülle und eGFP-markiertem RNP konnte gezeigt werden, dass zweifarbige, also umhüllte Virionen entlang der neuronalen Ausläufer von in vitro differenzierten und primären Neuronen transportiert wurden, wobei der retrograde Transport in den in vitro differenzierten Neuronen mit einer konstanten Durchschnittsgeschwindigkeit von 0,1 µm/sek und einer durchschnittlichen Transportlänge von 25 µm erfolgte.

The extracellular matrix (ECM) provides the structural frame for the development of tissues and organs. The ECM is bound by numerous membranous matrix-adhesion molecules and thereby triggers intracellular signals that control various cellular functions such as survival, polarity, proliferation and differentiation. Integrins represent an important family of ECM adhesion molecules which link the ECM with the intracellular actin-cytoskeleton. Integrin mediated adhesion structures also serve as important signaling platforms, although the integrin itself does not harbors any catalytic domains. Therefore integrin signaling depends on the recruitment of a number of cytoplasmic proteins that directly or indirectly bind to the short cytoplasmic integrin tails. During my PhD thesis I worked on three of these molecules, ILK, Kindlins and Palladin, and used the mouse as a model system to address their in vivo function. First, I investigated the role of integrin-linked kinase (ILK) in skeletal muscle. Loss of ILK expression in mice leads to peri-implantation lethality due to a cell polarization defect of the early embryo and abnormal actin accumulations. Studies in Caenorhabditis elegans and Drosophila melanogaster revealed an essential function for ILK in the attachment of actin filaments to the membrane of muscle cells and lack of ILK expression results in early lethality during embryogenesis. We generated mice with a skeletal muscle-restricted deletion of ILK that developed a mild, but progressive muscular dystrophy. This phenotype is predominantly restricted to myotendinous junctions (MTJs). Ultrastructural analyses showed muscle cell detachment from the basement membranes, and an accumulation of extracellular matrix. Endurance exercise training enhances the defect leading to disturbed subsarcolemmal myofiber architecture and an abrogation of the phosphorylation of Ser473 as well as Thr308 of protein kinase B (PKB)/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Second, I studied the expression and in vivo function of a further integrin- and actin- associated protein, palladin. Palladin belongs to the palladin/myotilin/myopalladin protein family. Palladin represents a phosphoprotein which plays an important role in cell adhesion and motility. Initially, I characterized the gene structure and the expression pattern of palladin. The palladin gene spans about 400 kb, with 25 exons and 3 alternative promoters resulting in at least three different isoforms (200 kDa, 140 kDa and 90-92 kDa) in mice. Using RT-PCR and in situ hybridizations of embryonic and adult tissues, I could show that the 200kDa isoform is predominantly expressed in heart and skeletal muscle. In contrast, the 140kDa isoform is expressed in various tissues and represents the major palladin isoform of the brain. The 90-92 kDa isoform is almost ubiquitously expressed with highest levels in tissues rich in smooth muscle, like bladder, uterus, small intestine and colon. The expression of the 200kDa isoform was characterized in more detail with a polyclonal antibody showing that this isoform localizes to the Z-discs of heart and skeletal muscle cells. In vitro differentiation experiments with a mouse myoblast cell line revealed an induction of the 200kDa isoform during myoblast fusion and differentiation suggesting that the biggest palladin isoform may serve as a molecular scaffold during myogenesis. Third, I specifically inactivated the largest palladin isoform in mice. Lack of the 200 kDa palladin isoform has no impact on the development, viability and fertility of mice. However ultrastructural analyses by transmission electron microscopy (TEM) showed a mild cardiac myopathy due to disintegration of myofibrils. In collaboration with the group of Olli Carpén, we generated palladin 200 kDa isoform/ myotilin double knockout mice. Myotilin is also expressed in heart and skeletal muscle. Ablation of both myotilin and palladin 200 kDa isoform in mouse revealed in addition to the mild cardiac myopathy a structural and functional impairment of skeletal muscle. Finally, I was also involved in the characterization of the expression and subcellular localization of a novel family of integrin associated proteins: the Kindlins. The Kindlin family consists of three members, Kindlin-1, -2 and -3. Mutations in Kindlin-1 cause a human disease, called Kindler Syndrome, which represents a skin blistering disease affecting the actin cytoskeleton of basal keratinocytes. Kindlin gene expression was first analyzed at the mRNA level by RT-PCR and Northern Blot studies. In situ hybridizations showed that Kindlin-1 is preferentially expressed in epithelia. Kindlin-2 is expressed in all tissues with highest levels in striated and smooth muscle cells. While both localize to integrin-mediated adhesion sites in cultured keratinocytes Kindlin-2, but not Kindlin-1, colocalizes with E-cadherin to cell-cell contacts in differentiated keratinocytes. In contrast, Kindlin-3 expression is restricted to hematopoietic cells. Using a Kindlin-3-specific antiserum and an EGFP-tagged Kindlin-3 construct, we could show that Kindlin-3 is present in podosomes, which are specialized adhesion structures of hematopoietic cells.

Fri, 8 Feb 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/8483/ https://edoc.ub.uni-muenchen.de/8483/1/Go_Stella.pdf Go, Stella Bettina Li-hsia ddc:500, ddc:590, Tierärz

Background: Pluripotent embryonic stem (ES) cells that can differentiate into functional cardiomyocytes as well as vascular cells in cell culture may open the door to cardiovascular cell transplantation. However, the percentage of ES cells in embryoid bodies (EBs) which spontaneously undergo cardiovascular differentiation is low (< 10%), making strategies for their specific labeling and purification indispensable. Methods: The human connexin 40 (Cx40) promoter was isolated and cloned in the vector pEGFP. The specificity of the construct was initially assessed in Xenopus embryos injected with Cx40-EGFP plasmid DNA. Stable Cx40-EGFP ES cell clones were differentiated and fluorescent cells were enriched manually as well as via fluorescence-activated cell sorting. Characterization of these cells was performed with respect to spontaneous beating as well as via RT-PCRs and immunofluorescent stainings. Results: Cx40-EGFP reporter plasmid injection led to EGFP fluorescence specifically in the abdominal aorta of frog tadpoles. After crude manual enrichment of highly Cx40-EGFP- positive EBs, the appearance of cardiac and vascular structures was increased approximately 3-fold. Immuno fluorescent stainings showed EGFP expression exclusively in vascular-like structures simultaneously expressing von Willebrand factor and in formerly beating areas expressing alpha-actinin. Cx40-EGFP-expressing EBs revealed significantly higher numbers of beating cardiomyocytes and vascular-like structures. Semiquantitative RT-PCRs confirmed an enhanced cardiovascular differentiation as shown for the cardiac markers Nkx2.5 and MLC2v, as well as the endothelial marker vascular endothelial cadherin. Conclusions: Our work shows the feasibility of specific labeling and purification of cardiovascular progenitor cells from differentiating EBs based on the Cx40 promoter. We provide proof of principle that the deleted CD4 (Delta CD4) surface marker-based method for magnetic cell sorting developed by our group will be ideally suitable for transference to this promoter. Copyright (c) 2008 S. Karger AG, Basel.

Zur Bekämpfung von genetisch bedingten Krankheiten werden oft Medikamente eingesetzt, die nur die Symptome bekämpfen, ohne aber die Ursache des Leidens zu eliminieren. Mit Hilfe der Gentherapie, so die Hoffnung, soll der Krankheits-verursachende Gendefekt durch therapeutische Fremdgene geheilt werden. In dieser Arbeit wurde eine auf EBV basierte Verpackungszellinie zur Herstellung von Genvektoren etabliert, welche unter Berücksichtigung aller derzeit bekannten Sicherheitsrisiken für eine Gentherapie optimiert wurde. Eine mögliche Anwendung für dieses EBV-basierte Gentransfersystem ist die Stimulierung von B-CLL-Zellen durch Expression des humanen CD40-Liganden. Dadurch sollen die Leukämiezellen einer Erkennung durch spezifische T-Zellen zugänglich gemacht werden. Für die Verwendung eines EBV-Genvektorsystems spricht unter anderem die hohe Effizienz der spezifischen Transduktion humaner B-Zellen, die große Fremdgen-Kapazität und die Fähigkeit zur latenten Infektion und daher langandauernden Genexpression. Zudem repliziert EBV episomal, modifiziert also nicht das Zellgenom. Allerdings ist EBV ein potentielles Tumorvirus. Daher wurden alle fünf bekannten Onkogene sowie der Transaktivator BZLF1 aus dem Helfergenom entfernt. Durch Deletion der Verpackungssignale wurde das Helfergenom so modifiziert, daß es nicht selbst in Virionen verpackt und freigesetzt werden kann. Die Verpackungseffizienz der Helferzellinie konnte durch FACS-Sortierung verbessert werden. Das EBV-Helfergenom wurde aus dieser Zellinie 293-VII+ reisoliert und seine Integrität durch PCR und Restriktionslängenvergleich bestätigt. Selbst bei provozierter Rekombination wurden von der Verpackungszelllinie 293-VII+ keine Virionen freigesetzt, die B-Zellen transformieren können. Ein weiterer Schwerpunkt dieser Arbeit war die Etablierung des therapeutischen hCD40L-tragenden Genvektors p2924 mit möglichst geringer Homologie zum Helfervirusgenom (TR und oriLyt als einzigen EBV-Sequenzen) und Verzicht auf Antibiotika-Selektionsmarker (stattdessen das nonsense suppressor-Transfer-RNA-Gen supF). Der bereits etablierte eGFP-tragende Genvektor p1933, welcher um etwa 6kb größer war und zusätzlich oriP trug, zeigte aber bessere Transfektionseigenschaften als p2924. Aus diesem Grund wurde unter anderem ein weiteres Genvektorplasmid konstruiert, bei welchem eGFP von p1933 durch hCD40L ersetzt wurde. Die Infektion bzw. Detektion von hCD40L auf B-CLL-Zellen war nur mit aufkonzentrierten Virusüberständen reproduzierbar, die mit diesem Plasmid hergestellt wurden. Allerdings trägt dieser Genvektor Amp als Selektionsmarker. Daher wurde zuletzt exemplarisch in dem eGFP-tragenden „großen“ Plasmid Amp durch supF ersetzt. Bislang wurden zur Propagierung von supF-Plasmiden Bakterienstämme verwendet, die die amber-Mutationen auf einem extrachromosomalen Plasmid enthielten. Um die einfache Gewinnung reiner Plasmidpräparationen zu ermöglichen, wurde auf der Basis von DH10B ein neuer Bakterienstamm mit chromosomaler amber-Mutation etabliert. Es wurde gezeigt, daß dieser Stamm sich zur antibiotikafreien Selektion und Produktion von supF-tragenden Plasmiden eignet. Somit stellt 293-VII+ eine optimierte Verpackungszelllinie dar, mit der EBV-basierte Genvektoren effizient hergestellt werden können, die sowohl etablierte B-Zelllinien als auch primäre B-Zellen transduzieren. Die erreichbaren Titer waren mit denen vergleichbar, die von der Verpackungszelllinie der ersten Generation (TR-2/293) produziert wurden. Die Produktion von Interferon- durch T-Zellen war erhöht, wenn sie mit B-CLL-Zellen stimuliert wurden, die zuvor mit Überständen aus verpackbaren, hCD40L-tragenden Vektoren nach Induktion des lytischen Zyklus transduziert wurden. Dieses Ergebnis lässt auf Aktivierung des Immunsystems in vivo hoffen. Ein völlig neuer Aspekt, der im Rahmen dieser Arbeit erstmalig beobachtet werden konnte, war der Übertrag von eGFP-Protein aus der Verpackungszelllinie in Rezipientenzellen. Alle Beobachtungen lassen auf einen spezifischen Transfer des fluoreszierenden Proteins aus dem Zytoplasma der Verpackungszelle auf die Oberfläche der B-Zellen durch Exosomen schließen. Experimente mit dem Modellantigen pp65 zeigten, dass auch dieses Protein direkt übertragen werden konnte und dadurch die Aktivierung von antigenspezifischen T-Zellen induzierte. In ähnlicher Weise konnten auch in einem reduzierten System die parentalen 293HEK-Zellen nach Transfektion mit Plasmiden für das EBV-Glykoprotein gp350/220 und das Antigen pp65 Überstände produzieren, die zu einer spezifischen Stimulation von T-Zellen führten. Diese Ergebnisse legen die zukünftige Entwicklung eines an EBV angelehnten Antigentransfersystems nahe, durch das mit Hilfe von B-Zellen als Stimulatoren eine spezifische T-Zellaktivierung erreicht werden kann.

Die normale Entwicklung von Embryonen nach somatischem Zellkerntransfer („somatic cell nuclear transfer“, SCNT) hängt unter anderem von der erfolgreichen Reprogrammierung und Aktivierung von Schlüsselgenen ab. Ein Beispiel ist das Gen für den Transkriptionsfaktor Oct4, der im frühen Embryo nachweisbar und als Marker für pluripotente Zellen gilt. Die in klonierten Mausembryonen häufig beobachtete abnormale Expression von Oct4 wird als eine mögliche Ursache für die hohen Verluste und schweren Fehlentwicklungen nach Kerntransfer diskutiert. Beim Rind liegt im Vergleich zur Maus und anderen Spezies die Erfolgsrate am höchsten. Daher ist die Untersuchung der Reprogrammierung von OCT4 nach SCNT in der frühen Embryogenese beim Rind von besonderem Interesse. Um Fragen der epigenetischen Reprogrammierung des Rindergenoms und der Rolle von OCT4 nach SCNT nachzugehen, wurden bovine fetale Fibroblasten stabil mit einem Oct4-EGFP-Reportergenkonstrukt transfiziert. In den unilokulär stabil transfizierten Zellen mit unauffälligem weiblichen Karyotyp war in Analogie zur Inaktivität des endogenen OCT4-Gens in differenzierten Zellklonen keine EGFP-Fluoreszenz nachweisbar. Das Anschalten der Oct4-EGFP-Expression nach SCNT entsprach weitgehend der nach in vitro Fertilisation beobachteten Aktivierung des endogenen OCT4-Gens. In SCNT-Embryonen mit weniger als neun Zellkernen wurde keine EGFP-Fluoreszenz nachgewiesen. In allen Embryonen mit mindestens 17 Zellkernen war das Oct4-EGFP-Reportergenkonstrukt aktiv, was darauf hindeutet, dass Blastomeren nach der vierten Zellteilung den Oct4-Promotor aktivierten. Mittels konfokaler Laser Scanning Mikroskopie wurde an zentralen optischen Schnitten die Intensität der EGFP-Fluoreszenz jedes Embryos gemessen. Im Vergleich mit Tag 4 SCNT-Embryonen war die EGFP-Fluoreszenz in Tag 6 Embryonen deutlich stärker mit erheblichen Unterschieden in der Expressionshöhe zwischen einzelnen Embryonen. Dabei zeigten Embryonen mit einer niedrigeren EGFP-Fluoreszenz im Vergleich zu Embryonen mit stärkerer EGFP-Fluoreszenz einen erheblich höheren Anteil an Zellkernuntergängen (kondensierte und fragmentierte Zellkerne). 34 Tage nach dem Transfer von EGFP-exprimierenden Embryonen auf Empfängertiere wurden drei lebende und morphologisch unauffällige Feten gewonnen. In Fibroblasten, die aus diesen Feten isoliert wurden, war das Reportergenkonstrukt, analog zur normalen Inaktivierung des endogenen OCT4-Gens in differenzierten Zellen, inaktiviert. In SCNT-Embryonen aus den Oct4-EGFP-transgenen Fibroblasten dieser zweiten Generation („second round“ SCNT) wurde das Reportergenkonstrukt erneut regelmäßig aktiviert wie in den SCNT-Embryonen vom Ausgangszellklon („first round“ SCNT). Die Herstellung stabil transfizierter boviner fetaler Fibroblasten mit einer unilokulären Integration des Oct4-EGFP-Reportergenkonstruktes stellt eine wichtige Basis für ein breites Spektrum experimenteller Ansätze zur Aufklärung grundlegender Mechanismen nach Kerntransfer beim Rind dar.

Lentiviral transduction of oocytes or early embryos is an efficient strategy to generate transgenic rodents and livestock. We evaluated laser-based microdrilling (MD) of the zona pellucida, which is a physical barrier for viral infection, and subsequent incubation in virus suspension as a new route for lentiviral transgenesis in bovine. Lentiviral vectors carrying an eGFP expression cassette were used to transduce oocytes or zygotes after MD as compared to the established subzonal virus injection technique (MI). The type of manipulation (MD vs. MI) did not affect cleavage rates, but had a significant effect on blastocyst rates (p < 0.001). MI of virus or sham-MI (buffer) resulted in higher blastocyst rates as compared to MD, both in the oocyte and zygote treatment groups. The latter exhibited higher rates of early cleavage (p < 0.05) and blastocyst rates (p < 0.01). The proportion of eGFP expressing blastocysts was higher after infection of oocytes (MD: 44 ± 9%; MI: 67±8%) than after infection of zygotes (MD: 26 ± 8%; MI: 26 ± 9%). Overall efficacy (eGFP-positive blastocysts per treated oocytes or zygotes) was highest after MI of oocytes (18 ± 2%). Our study demonstrates the feasibility of laser-assisted lentiviral gene transfer into bovine oocytes and zygotes. However, further optimization of the procedure is required, mainly to reduce the incidence of polyspermy after MD of oocytes and to eliminate negative effects of MD on early embryonic development.

The development of relevant cellular model systems for colorectal cancer is of utmost importance for an improved in vitro assessment of therapeutic strategies against colorectal cancer. Recently published low passage colon cancer cell lines that closely reflect the characteristics of the respective parental in vivo tumor cells represent very promising cell culture models and were therefore used for the investigations in the present thesis. To provide an in vitro model system that also recapitulates the three-dimensional structure of in vivo tumors, these low passage cell lines were cultivated as multicellular spheroids. Compared to monolayer cultures the multicellular spheroids exhibited a wide variety of changes in their expression patterns. The differential expression includes proteins that are involved in growth signaling (15-hydroxyprostaglandin dehydrogenase), protein biosynthesis (acidic ribosomal protein P0), and regulation of the cyto- or nucleoskeleton (acidic calponin and LMNA protein). These proteins were identified by 2D electrophoresis and subsequent MALDI-TOF mass spectrometry. Both methods were established in the lab in the context of this work. Chemotherapy with 5-fluorouracil (5-FU) represents the traditional treatment of colorectal cancer. However, in many patients the efficiency of this therapeutic strategy is often limited by the development of chemoresistance against 5-FU. Therefore, it was an aim of this thesis to detect novel proteins involved in 5-FU chemoresistance that were previously not ascribed to resistance against this chemotherapeutic drug. A chemoresistant subline of a colon cancer cell line was generated by long-term treatment with 5-FU and served as a model for the investigation of 5-FU chemoresistance. This subline exhibited resistance against both 5-FU-induced inhibition of proliferation and apoptosis. Differences in the expression of cytokeratin 18, heat shock protein 27 and aldehyde dehydrogenase 1B1 between the chemoresistant subline and parental cells were detected by 2D electrophoresis. These findings imply that the cytoskeleton plays a role in the development of chemoresistance against 5-FU. Furthermore, processes located to the mitochondria seem to be involved in this resistance, since heat shock protein 27 and aldehyde dehydrogenase 1B1 are associated with this subcellular organelle. The biological relevance of the findings made in the present PhD thesis has to be determined in further studies. Gene therapy represents a promising alternative strategy for the treatment of colorectal cancer. A novel nonviral gene transfer system was developed by combination of DNA with the polycation PEI25br and the cationic lipids DOCSPER or DOSPER to form lipopolyplexes. These lipopolyplexes enabled enhanced gene transfer in vitro and are promising for in vivo applications, since the established lipopolyplexes preserved their small size at physiological conditions; a property essential for a successful in vivo application. Furthermore, the lipopolyplexes exhibited the capability to efficiently transfect three-dimensional multicellular spheroids. The potential of lipopolyplexes for therapeutic applications was further increased by the utilization of the artificial promoter CTP4 which enables highly specific gene expression in cancer cells with mutations in the Wnt signaling pathway by transcriptional targeting. In addition to its high specificity, this promoter enabled high gene expression levels that were comparable to expression levels obtained by the strong, but unspecific CMV promoter. The efficiency of the CTP4 promoter was demonstrated in seven low passage colon cancer cell lines and also in multicellular spheroids. The transcriptional targeted lipopolyplexes not only enabled high tumor specific expression of reporter genes like luciferase or EGFP but also the expression of a therapeutic gene, interleukin-2 (IL-2). Furthermore, tumor specific expression of cytotoxic protease 2A in combination with IL-2 was possible by using a novel bicistronic construct. The expression of the rhinoviral protease 2A led to efficient reduction of overall cap-dependent gene expression levels and therefore also the proliferation of the transfected cells, while continued IL-2 expression was guaranteed by an IRES element enabling cap-independent gene expression in the presence of protease 2A. In summary, the present results provide a promising basis for the development of novel potent strategies in the treatment of colorectal cancer.

Die chronische lymphatische Leukämie ist die häufigste Leukämie im Erwachsenenalter in den westlichen Ländern. Erkenntnisse der letzten Jahre haben das Spektrum verfügbarer Therapien deutlich erweitert, kurativ ist bisher nur die allogene Knochenmarkstransplantation. Im Blut der betroffenen Patienten treten die Zellen des malignen Klons in Kontakt mit autologen Immuneffektorzellen. Gentherapeutische Strategien basierend auf dem Transfer immunstimulatorischer Moleküle eröffnen deshalb neue Perspektiven für die Therapie der B-CLL. In der vorliegenden Arbeit wird erstmals die Eignung eines Helfervirus-freien EBV-Genvektorsystems für den Gentransfer in primäre B-CLL-Zellen beschrieben. Durch Optimierung der Vektorverpackung für ein eGFP kodierendes Plasmid konnte gezeigt werden, dass sich mit Hilfe einer Helfervirus-freien Verpackungszelline infektiöse Titer bis zu 2 x 106 infektiöse Partikel/ml generieren lassen. Das untersuchte Vektorsystem eignet sich für einen effizienten Gentransfer in primäre B-CLL-Zellen. Auf eine CD40L-Stimulation, wie sie zur Verbesserung der Transfereffizienz von Adenoviren zur Anwendung kommt, kann dabei verzichtet werden. Der Gentransfer lässt sich mit Hilfe eines monokonalen Antikörpers gegen CD21 weitgehend neutralisieren. In Kontrollexperimenten mit Überständen aus der Verpackung TR-deletierter Genvektorplasmide sinkt die Gentransferrate auf Werte nahe der Nachweisgrenze. Nach Verpackung des therapeutischen Moleküls CD40L sinkt die Transfereffizienz gegenüber dem eGFP kodierenden Genvektor und vermehrt unspezifische Effekte werden beobachtet. Zu den charakteristischen Eigenschaften EBV abgeleiteter Genvektoren zählen ein Tropismus für B-Lymphozyten und eine grosse Verpackungskapazität, die den Transfer von Gensequenzen bis zu einer Grösse von 160 kb erlaubt. Als essentielle EBV-Elemente enthalten die Genvektoren nur die Verpackungssignale TR und den lytischen Replikationsorigin oriLyt. Zukünfte Entwicklungen des Vektorsystems haben zum Ziel das Risiko von Rekombinationsereignissen zu reduzieren, die zur Freisetzung replikationsfähiger Virusmutanten oder potentiell onkogener viraler Gene führen könnten.

In situations where well-established approaches such as surgery, radiation therapy and chemotherapy fail to help cancer patients, immunotherapy has the potential to be an effective alternative. Tumour cells can sometimes be distinguished from corresponding normal cells due to their expression of tumour-associated antigens (TAAs), most of which are unaltered self-molecules. These molecules must be presented to the immune system in the context of danger in order to achieve their specific recognition. If dendritic cells (DCs), the most potent professional antigen-presenting cells, are loaded with RNA, they will translate the RNA into protein, process the protein into peptides and present the peptides within MHC molecules (pMHC) on their surface to cytotoxic T-lymphocytes (CTLs) and T-helper cells in a stimulatory manner. These effector cells can, in turn, recognise tumour cells. The goal of these studies was to find optimal conditions for producing a DC-based vaccine for cancer patients using TAAs in the form of RNA. The studies were designed to quantitate RNA transfer into DCs, to determine the intracellular stability of transfected RNA in DCs and to analyse the kinetics of protein expression and the generation of functional pMHC ligands that could activate effector memory CTLs. Simultaneous activation of CTLs with specificities for different antigens minimises the potential for tumour escape through immune selection of tumour variants showing loss of individual antigens. Thus, generation of multiplex pMHC ligands for CTLs may improve clinical efficiency. On the other hand, peptide competition for MHC molecules within the DC may limit pMHC ligand generation. This central immunological question was addressed by comparing DCs loaded with total cellular tumour RNA, amplified total cellular tumour mRNA and pools of defined single-species tumour-antigen cRNAs versus individual single-species tumour-antigen cRNAs for their capacity to display various pMHC ligands and activate CTLs of corresponding specificities. Experiments performed with RNA encoding the enhanced green fluorescence protein (EGFP), a reporter protein, showed that the highest efficiency of RNA transfection into DCs was achieved with electroporation, reaching levels of 90% positive cells. The fact that mature DCs expressed more EGFP than immature DCs suggests that this stage of DC maturation will be optimal for vaccine development. Importantly, electroporation and RNA transfer did not alter the expression of antigen-presenting and co-stimulatory molecules on the surface of DCs. The melanoma model was chosen for extensive analyses because its characterisation at the cellular and molecular levels has made it a very informative model for understanding cancer immunity. In addition to total cellular melanoma RNA, single-species cRNAs were used encoding the melanoma-associated antigens, tyrosinase, Melan-A and CDK4-R24C. Antigen presentation was detected with the help of effector memory CTL clones specific for each of these antigens. The CTL stimulatory capacity of RNA-transfected DCs was higher if they were allowed one day to recuperate from electroporation and to produce pMHC complexes. Tyrosinase cRNA dose-finding showed that more RNA would indeed result in higher stimulatory capacities of transfected DCs. Kinetics of tyrosinase cRNA degradation, similar to kinetics of EGFP cRNA degradation, revealed that the amount of transfected RNA rapidly decreased inside the DCs within 1.5 hr after electroporation. The smallest decrease was observed with the highest amount of RNA applied in electroporation. The kinetics of RNA degradation and protein half-life will be important parameters to consider in defining the right time-frame for T-cell activation by engineered DCs. When reverse transcription PCR (RT-PCR) was performed with total cellular melanoma RNA samples to generate amplified mRNA, the Melan-A, tyrosinase and CDK4/CDK4-R24C message was amplified 62-fold, 24-fold and 2-fold, respectively. These differences likely reflect variations in the expression levels of the corresponding antigen message in melanoma cells from which the RNA was isolated. Approximately 17240-fold more CDK4-R24C message, at least 500-fold more tyrosinase message and at least 480-fold more Melan-A message was found in single-species cRNAs when the same masses of single-species cRNA and amplified melanoma mRNA samples were compared. This explained why electroporation of single-species cRNAs into DCs yielded the highest DC stimulatory capacities. Combinations of tyrosinase, Melan-A and CDK4-R24C cRNAs were studied for their capacity to induce satisfactory levels of T-cell stimulation when presented by DCs. Here it was demonstrated that antigen competition was not a critical factor, since CTL responses to pooled RNAs were not inhibited even though competition for MHC class I molecules may have occurred within the DCs. DCs also developed CTL stimulatory capacities, but at much lower levels, using amplified melanoma mRNA. Two antigen-specific CTL clones displayed higher reactivities upon exposure to pMHC produced naturally by RNA-transfected DCs than to synthetic peptides pulsed onto DCs. In one case, this could be explained by a post-translational modification of the peptide, which normally occurs within cells. Since this particular modification was not represented in the synthetic peptide, which was chosen from the protein sequence, the synthetic peptide was not well recognised. This demonstrated that the use of RNA technology eliminates the need to know the correct sequences of immunogenic peptides. Thereby, DCs are better than scientists at choosing antigens and their epitopes for presentation to T-cells. These data provided a better understanding of antigen presentation by DCs based on the use of RNA, giving insight into antigen competition and paving the way for the use of pooled RNAs of defined species for the development of a multiplex vaccine. They also allowed a precise protocol for efficient T-cell activation to be defined. Further experiments will demonstrate whether quantitative differences detected in antigen presentation between DCs loaded with total cellular tumour RNA and amplified total cellular tumour mRNA versus single-species tumour-antigen cRNAs have an impact on de novo T-cell priming in vitro and in vivo.

Human artificial chromosomes (HACs) were generated by transfer of telomerized PAC constructs containing alpha satellite DNA of various human chromosomes. To monitor which cells took up constructs and subsequently formed stable clones under blasticidin S (BS) selection, a CMV/EGFP expression cassette was inserted into a HAC construct based on chromosome 5 alpha satellite DNA (142 kb). Lipofection into HT1080 cells resulted in a small proportion of cells exhibiting bright green fluorescence on day 1. Areas containing such early green cells were marked, and plates monitored over 2 weeks. In only one out of 41 marked areas, a viable clone developed. In the remaining 40 areas, the green cells ceased division at 1-8 cells. In contrast, outside the marked areas, 16 stable clones formed which did not exhibit green fluorescence during the first cell divisions, but all cells of each became green around day 4 -6. Fluorescence in situ hybridization (FISH) analysis of isolated clonal lines demonstrated low copy HAC formation without integration. We conclude that transient expression of an EGFP marker on HAC DNA is not a suitable means for the identification of the proportion of transfected cells which are capable of forming viable clones. One explanation could be that the high copy number required to consistently detect transient EGFP expression (Schindelhauer and Laner, 2002) impairs viability and clone formation. Copyright (C) 2004 S. Karger AG, Basel.

Die HCN Kanäle spielen als molekulare Basis des Ih Stroms eine bedeutende Rolle bei der Entstehung rhythmischer Erregungen. Die HCN Familie der Säugetiere besteht aus vier Mitgliedern (HCN1-HCN4), die in unterschiedlichem Ausmass in verschiedenen Regionen von Herz und Gehirn exprimiert werden. Im Rahmen der vorliegenden Arbeit wurden die Bildung von Heteromeren zwischen den einzelnen Isoformen, die Glycosylierung der Kanäle und deren Funktion, die Modulation durch cAMP, die Aktivierung in Bezug auf Kinetik und Spannungsabhängigkeit, sowie die Rolle von HCN Kanal-Genen im Rahmen von genetischen Erkrankungen untersucht. Sowohl im Mausgehirn wie auch in HEK Zellen können die HCN Kanal-Untereinheiten miteinander interagieren. Im Mausgehirn wurde die Heteromerisierung von HCN1 und HCN2 durch Ko-Immunpräzipitation nachgewiesen. Durch konfokalmikroskopische Studien an EGFP- bzw. RFP-markierten HCN Isoformen in HEK Zellen konnte für alle HCN-Zweierkombinationen ausser mHCN2 mit mHCN3 eine Kolokalisation im Bereich der Plasmamembran beobachtet werden. Bestätigt wurden diese Ergebnisse durch Ko-Immunpräzipitation. Mit Hilfe von elektrophysiologischen Untersuchungen konnte verifiziert werden, dass mHCN2 und mHCN3 nicht interagieren. Die HCN Kanäle werden in vivo (Mausgehirn) und in vitro (HEK Zellen) N-glycosyliert. Am Beispiel des mHCN2 wurde die physiologische Relevanz dieser posttranslationalen Modifikation untersucht. Ein mutanter, unglycosylierter mHCN2 Kanal wird nicht mehr zur Plasmamembran transportiert und generiert keinen charakteristischen Einwärtsstrom mehr. Nach Koexpression mit hHCN4 ist die Membranständigkeit dieser Mutante wieder gegeben, sowie auch die Kolokalisation der beiden Kanäle. Im Gegensatz hierzu konnte mHCN3 den unglycosylierten mHCN2 nicht zur Zellmembran transportieren, da zwischen den beiden Kanälen ganz offensichtlich keine Bindung bestehen kann. Die für die Bindung von cAMP an den mHCN2 Kanal essentielle Aminosäure Arginin 591 wurde durch gerichtete Mutagenese und Patch-Clamp-Untersuchungen identifiziert. Der Ersatz des Arginin 591 führt zu mHCN2 Kanälen, die bei Anwesenheit von cAMP nicht mehr wie der Wildtyp Kanal eine Verschiebung der halbmaximalen Aktivierung zu positiveren Potentialen zeigen. Durch elektrophysiologische Charakterisierung von verkürzten mHCN2 Kanälen und Chimären aus mHCN1 und mHCN2 konnten den Unterschieden in Aktivierungskinetik und Spannungsabhängigkeit der HCN Isoformen konkrete Kanalteile zugeordnet werden. Für die Geschwindigkeit der Aktivierung sind die Kernregion und ein Stück des C-Terminus verantwortlich, während der C-Terminus die Spannungsabhängigkeit bestimmt. Die genomische Analyse des HCN2 von Cayman Ataxie-Patienten, die zerebrale Dysfunktionen ähnlich denen von HCN2-knockout Mäusen zeigen, ergab keine Unterschiede im Vergleich zu einem gesunden Probanden. Eine Mutation im HCN2 Gen als Ursache der Cayman Ataxie kann somit ausgeschlossen werden, es besteht jedoch die Möglichkeit einer Störung der Proteinsynthese oder posttranslationaler Modifikationen. Auch bei zwei Patienten, die an familiär bedingtem Sick Sinus Syndrom leiden, konnte die Ursache der Erkrankung nach genomischer Sequenzanalyse nicht durch einen Defekt des HCN2 Gens oder des HCN4 Gens erklärt werden.

Generation of Transgenic Mice to Evaluate Promoter Activity and Specificity of two Human Endogenous Retrovirus Long Terminal Repeats Human Endogenous Retrovirus Long Terminal Repeats (HERV-LTRs) comprise 1.8% of the human genome (52.7 Mb). These sequences contain all the signal structures necessary for the regulation of gene transcription, such as promoters, enhancers and transcription factor binding sites. There is evidence that HERV-LTRs regulate gene expression in tissue-specific manner. This potential could be used to drive the expression of therapeutic genes, delivered by retroviral vector systems, in a safe and efficient manner. The HERV-H-H6 LTR and the HERV-L LTR were chosen for the generation of transgenic mice. Their promoter activity and specificity had prior been tested in a luciferase expression vector in vitro (Schoen et al., 2001). HERV-L was cloned into a luciferase expression vector and HERV-H-H6 was inserted into a enhanced green fluorescent protein (EGFP) expression vector. Transgenic mice were generated by DNAmicroinjection into pronuclei of zygotes. One pBL-HERV-L transgenic line and four pEGFP-HERV-H-H6 transgenic lines were established and analyzed. While the HERV-L promoter was not active in transgenic animals, pEGFP-HERV-H-H6 was expressed in gonads of mice of two transgenic lines. As only a single, non-expressing transgenic line was available, HERV-L promoter activity and specificity could not be evaluated. Additional transgenic lines have to be established. Expression level and pattern of the HERV-H-H6 promoter indicate specificity for gonad tissue. Whether the HERV-H-H6 promoter activity is linked to steroid production in cells remains to be clarified. Evaluating promoter activity in transgenic mice in two different expression vectors is not exclusively about the promoters, but also involves knowledge about the reporter genes. Advantages and limits of current applications of both luciferase and EGFP (with focus on the EGFP gene) are described in REVIEW OF THE LITERATURE. The conjunction of EGFP with the HERV-H-H6 promoter is to be seen critically, as all published methods for detection of EGFP in mice are described with EGFP linked to strong promoters. Problems like autofluorescence in fluorescence microscopy might be encountered when weaker promoters, such as HERV-LTRs, drive EGFP expression.

Programmierter Zelltod oder Apoptose ist essentiell für die Entwicklung und Homöostase mehrzelliger Organismen. Störungen in der Regulierung dieser Prozesse können zu zahlreichen Erkrankungen führen, unter anderem zu Autoimmunerkrankungen und Krebs. In den letzten Jahren konnte in zahlreichen Arbeiten gezeigt werden, daß Mitochondrien eine wichtige Rolle bei der Steuerung der Apoptoseprozesse spielen. So wird Cytochrom c, ein Protein aus dem mitochondrialen Intermembranraum, durch einen pro-apoptotischen Stimulus als ein Caspase-Aktivierungsfaktor ins Cytosol freigesetzt. In der vorliegenden Arbeit wird die initiale Charakterisierung eines neuen murinen Proteins (mDAP-3) beschrieben. mDAP-3 wurde identifiziert bei dem Versuch molekulare Marker der Nierenentwicklung mit Hilfe einer modifizierten „differential display“ Polymerase Kettenreaktion zu finden. Die 1,7 kb große mDAP-3 mRNA kodiert für ein ca. 45 kDa großes Protein, das als funktionelles Motiv eine ATP/GTP-Bindungsstelle (P-Loop) besitzt. Die Analyse der Aminosäurensequenz von mDAP-3 ergab eine 81 %ige Identität zu dem humanen DAP-3 (Death Associated Protein 3), einem positiven Apoptose Mediator. Northern-Blot-Analyse von 20 µg Gesamt-RNA aus 11 Organen adulter Mäuse zeigte eine abundante mDAP-3 Expression in Niere, Herz, Leber, Thymus, Muskel, Milz, Darm und Bauch, mit einem Expressionsmaximum in Testis. Eine geringe bis fast fehlende Expression konnte in der Lunge und im Ovar gezeigt werden. Die Überexpression eines mDAP-3/EGFP Fusionproteins in murinen Mesangialzellen (MMC) führte zu einer Apoptoseinduktion bei 27,6% ± 2,6% (n=3) der Zellen gegenüber einer Apoptose Inzidenz von 11,9% ± 2,8% bei MMCs die mit einem Kontrollvektor transfiziert wurden. Die pro-apoptotische Funktion von mDAP-3 ist dabei abhängig von der Funktionalität des P-Loops. Die Überexpression eines P-Loop mutierten mDAP-3/EGFP Fusionproteins führte zu keiner Apoptoseinduktion. Sowohl das murine als auch das humane DAP-3 bleiben während der Apoptose intra-mitochondrial und werden nicht in das Cytosol freigesetzt. Für die Untersuchung der intrazellulären Lokalisation von mDAP-3 wurde ebenfalls das mDAP-3/EGFP-Fusionsprotein verwendet. Murine-Tubuluszellen (MTC) zeigten nach Transfektion mit dem Fusionsprotein ein punktiertes Signal im Fluoreszenz-Mikroskop. Im Gegensatz dazu zeigten Zellen nach Transfektion mit dem EGFP-Expressionsvektor alleine das für EGFP typische diffuse, zytosolische Fluoreszenzsignal. Sowohl im Fluoreszenz-Mikroskop als auch im konfokalen Laser-Scann-Mikroskop lokalisierte mDAP-3/EGFP zusammen mit einem mitochondrialen Farbstoff, jedoch nicht mit einem lysosomalen. Diese Ergebnisse weisen auf eine mitochondriale Lokalisation von mDAP-3 hin. Zellfraktionierungen bestätigten die mitochondriale Lokalisation von mDAP-3. Das endogene mDAP-3 Protein konnte nur in der mitochondrialen Fraktion, nicht jedoch in der endoplasmatischen Reticulum- oder der zytosolischen-Fraktion, nachgewiesen werden. Proteinase-K-Behandlung isolierter Mitochondrien führte zu keiner Reduktion der mDAP-3 Proteinmenge im Western-Blot. Dies ließ auf eine intra-mitochondriale Lokalisation von mDAP-3 schließen. Digitonin-Behandlung isolierter Mitochondrien zeigte eine Freisetzung von mDAP-3 aus den Mitochondrien bei relativ hohen Digitonin Konzentrationen (0,3%). Ganz im Gegensatz dazu wurde Cytochrom c bereits bei 0,075% Digitonin freigesetzt. Diese Daten weisen auf eine mDAP-3 Lokalisation in der mitochondrialen Matrix hin. Da die DAP-3 Proteinfamilie in Eukaryonten sehr konserviert ist und auch in nichtapoptotischen Organismen wie S. cerevisiae vertreten ist, muß DAP-3 eine weitere Funktion neben der pro-apoptotischen besitzen. Um die Rolle von DAP-3 näher zu definieren wurde eine Disruption des mDAP-3 Hefe-Orthologs YGL129c (yDAP-3) durchgeführt. Die yDAP-3 Nullmutanten zeigten keinen Wachstumsverlust auf nicht fermentierbaren Kohlenstoffquellen wie Glycerol oder Lactat. Dies deutet darauf hin, daß yDAP-3 für die mitochondriale Atmung nicht zwingend notwendig ist. Allerdings zeigten Hefen bei einer yDAP-3 Disruption einen signifikanten und progressiven Verlust ihrer mitochondrialen DNA (mtDNA) (46% in ∆yDAP-3 vs. 3% im Wildtyp). Dieser Verlust der mtDNA, der auf eine Funktion von yDAP- 3 für die mitochondriale Biogenese hinweist, ließ sich durch eine Transfektion der ∆yDAP-3 Hefen mit dem murinen DAP-3 teilweise verhindern. Damit konnte bewiesen werden, daß die Funktion, die DAP-3 für die Biogenese der Mitochondrien ausübt, unter Eukaryonten konserviert ist. Diese Daten identifizieren mDAP-3 als einen der ersten pro-apoptotischen Faktoren der mitochondrialen Matrix. Weiterhin kann eine duale Funktion für die Mitglieder der DAP-3 Proteinfamilie postuliert werden. Zum Einen spielen sie eine wichtige, evolutionär konservierte Rolle für die Biogenese von Mitochondrien, zum Anderen besitzen sie in mehrzelligen Organismen eine zusätzliche pro-apoptotische Funktion.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.20.050815v1?rss=1 Authors: Jiang, H., Pederson, S. M., Newman, M., Dong, Y., Lardelli, M. Abstract: PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer's disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross pigmentation defects, arguing against significant loss of {gamma}-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5' mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins that obey the "reading frame preservation rule" of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions. Copy rights belong to original authors. Visit the link for more info