82 research outputs found
Chemical mutagenesis of mouse embryonic stem cells
erste Seiten (Inhalt u.a.)
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Einleitung
4- 21
Methoden und Methodenetablierung
22- 39
Ergebnisse
40- 83
Diskussion
84- 99
Anhang
100-114Um die Funktionen der menschlichen Gene aufzuklären, sind Studien an
Modellorganismen essentiell. Das wichtigste Modelltier für den Menschen ist
die Maus. Mausmutanten sind bei der Aufklärung von Genfunktionen von hoher
Bedeutung, doch haben sämtliche etablierte Ansätze zur Gendisruption ihre
spezifischen Nachteile. Angesichts der Vielzahl an Genen unbekannter Funktion,
die durch großangelegte Sequenzierungsprojekte offenbart wurden, gibt es
allgemein einen Bedarf an effizienten und zielgerichteten Ansätzen zur
Produktion von Mausmutanten. Die klassische Methode, Gene in Mäusen
auszuschalten, verläuft über die Mutagenisierung der Keimbahn mit Hilfe
mutationsauslösender Agenzien. Die Generierung von Mausmutanten erfolgt dort
nach dem Zufallsprinzip. Demgegenüber bietet die Gendisruption in embryonalen
Stammzellen (ES-Zellen) den Vorteil, positive Klone in vitro zu selektieren
für die zielgerichtete Produktion von Mutanten. Durch die
sequenzunspezifische, chemische Mutagenesierung einer Population embryonaler
Stammzellen können statistisch sämliche Gene im Mausgenom getroffen werden.
Die in separate Proben zu verteilenden Klone könnten dann nach Mutationen in
interessierenden Genen durchmustert werden, um anschließend die selektive
Produktion von Mausmutanten zu ermöglichen. Gegenstand dieser Arbeit ist die
Etablierung eines solchen Ansatzes. Dazu wurden zwei Agenzien,
4,5',8-Trimethylpsoralen (TMP) und N-Ethyl-N-nitrosoharnstoff (ENU), in ihren
Eigenschaften als chemische Mutagene für ES-Zellen charakterisiert. Es stellte
sich heraus, dass sich das potenzielle Deletionsmutagen TMP wegen seiner
geringen Mutagenität und der Heterogenität induzierter Mutationen nicht als
Mutagenisierungsagenz für die Generierung eines ES-Zell-Klonarchivs eignet.
Das Punktmutagen ENU hingegen erfüllte die Voraussetzungen hoher Mutagenität
und Homogenität des Mutationsspektrums. Es wurden anschließend verschiedene
Strategien zur Identifizierung unbekannter Mutationen evaluiert, wobei die
Detektion von Exondeletionen auf Transkriptebene die weitaus höchsten
Ausdünnungen von Mutationen erlaubte. Da Spleiß-Mutationen einen signifikanten
Anteil am ENU-Mutationsspektrum ausmachten, wurde eine rund 40.000 Klone
umfassende Bibliothek erstellt mit der Absicht, hochgradig gepoolte cDNA-
Proben PCR-basiert nach Spleiß-Mutationen zu durchmustern. Am Beispielgen Kit
sollte die Machbarkeit dieses Ansatzes demonstriert werden. Es konnten zwei
Klone mit Exondeletion im Kit-Transkript isoliert werden. Mit einem der beiden
Klone (Deletion von Exon 18) gelang die Transmission der Mutation durch die
Keimbahn. Die heterozygote Mausmutante zeigte einen für Null-Mutationen
charakteristischen Phänotyp.Model organisms play an essential role for studying the functions of human
genes. The most important model for the human is the laboratory mouse. Mouse
mutants present valuable tools for deciphering mammalian gene functions. The
established methods for disrupting gene functions, however, all have their
specific shortcomings. Facing large numbers of genes with unknown functions
there is a general need for efficient methodology to produce mouse mutants in
a directed fashion. Classically, knock-out mice are produced at random using
chemical mutagenesis to treat whole animals. In contrast, inactivating genes
in embryonic stem (ES) cells bears the advantage to select for positive clones
in vitro enabling a gene-driven production of mutants. By chemically
mutagenizing a set of ES cells virtually any gene in the mouse genome may be
affected. Generating a library of statistically mutated clones would therefore
allow to screen for mutations in a particular gene of interest. Mouse mutants
could then selectively be generated from clones with mutations identified in
that gene. The present work aims at establishing such an approach. To this
end, the properties of two mutagens, 4,5',8'-trimethylpsoralen (TMP) and
N-ethyl-N-nitrosourea (ENU), were characterized in ES cells. TMP appeared to
be inefficient as a mutagen and showed a heterogeneous spectrum of induced
mutations. In contrast, ENU proved to be highly effective. Interestingly, a
significant proportion of its mutational spectrum was presented by exon
deletions at the mRNA level. Moreover, comparing different PCR-based mutation
detection strategies revealed that the detection of deletions was most
sensitive. Therefore, a library of approximately 40,000 clones was generated
aiming at the identification of splice mutations. As a proof of concept,
highly pooled cDNA samples were screened for mutations that lead to exon
deletions in the Kit transcript. Two splice mutant clones could be isolated.
One of these, lacking exon 18, was successfully employed to transmit its
mutation through the mouse germline. The heterozygous mutant displayed a
specific phenotype resembling that of a dominant null mutation
The PluriNetWork: An Electronic Representation of the Network Underlying Pluripotency in Mouse, and Its Applications
BACKGROUND: Analysis of the mechanisms underlying pluripotency and reprogramming would benefit substantially from easy access to an electronic network of genes, proteins and mechanisms. Moreover, interpreting gene expression data needs to move beyond just the identification of the up-/downregulation of key genes and of overrepresented processes and pathways, towards clarifying the essential effects of the experiment in molecular terms. METHODOLOGY/PRINCIPAL FINDINGS: We have assembled a network of 574 molecular interactions, stimulations and inhibitions, based on a collection of research data from 177 publications until June 2010, involving 274 mouse genes/proteins, all in a standard electronic format, enabling analyses by readily available software such as Cytoscape and its plugins. The network includes the core circuit of Oct4 (Pou5f1), Sox2 and Nanog, its periphery (such as Stat3, Klf4, Esrrb, and c-Myc), connections to upstream signaling pathways (such as Activin, WNT, FGF, BMP, Insulin, Notch and LIF), and epigenetic regulators as well as some other relevant genes/proteins, such as proteins involved in nuclear import/export. We describe the general properties of the network, as well as a Gene Ontology analysis of the genes included. We use several expression data sets to condense the network to a set of network links that are affected in the course of an experiment, yielding hypotheses about the underlying mechanisms. CONCLUSIONS/SIGNIFICANCE: We have initiated an electronic data repository that will be useful to understand pluripotency and to facilitate the interpretation of high-throughput data. To keep up with the growth of knowledge on the fundamental processes of pluripotency and reprogramming, we suggest to combine Wiki and social networking software towards a community curation system that is easy to use and flexible, and tailored to provide a benefit for the scientist, and to improve communication and exchange of research results. A PluriNetWork tutorial is available at http://www.ibima.med.uni-rostock.de/IBIMA/PluriNetWork/
Large-Area RPE Removal by Microsecond Laser followed by hiPS-RPE transplantation
Cell therapeutics for AMD were often implanted regardless of RPE status in the target zone. This may result in RPE multilayering. Here we study a novel laser to remove RPE without collateral damage prior to RPE implantation to encourage better subretinal integration. Pigment rabbits (n=24) were immunosuppressed with Sirolimus, Doxycyclin and Minocyclin. Using a SLO/ OCT (Heidelberg Engineering) extended with a prototype laser (Meridian Medical; wavelength: 532 nm; pulse duration, 8 µs), a large area of RPE was selectively removed in 19 rabbits. Animals without laser lesions served as controls (n=5). A 25 gauge vitrectomy (Geuder) with removal of posterior hyaloid membrane was performed thereafter. Human iPS-RPE (1000 cells/ µl) were manually injected using a 100 µl syringe (Hamilton) connected to a 38G cannula (MedOne) into the RPE laser lesion, or over healthy RPE in controls, monitored by intraoperative OCT imaging (RESCAN 700, Zeiss). In vivo follow up/ retinal imaging was up to 12 weeks including fluorescein and indocyanine angiography, as well as SD-OCT (Spectralis ®, Heidelberg Engineering). Representative RPE laser wounds exhibited mild late phase FA& ICGA leakage, without abnormal outer retinal or choroidal hyperreflectivity on OCT. By contrast, lesions with earlier leakage on FA/ ICGA showed beam-sized outer retinal hyperreflectivity on OCT, suggesting coagulation. The size of the RPE wounds was typically 10-12mm2.iOCT demonstrated in an immediate and directed spread of the bleb retinal detachment (bRD) within the lasered zone. By contrast, bRDs performed over non-lasered RPE raised slower with a circular spread. Subretinal injection ranged from 5-70µl, with lesser volumes/ larger bRDs areas over lasered regions.At 6 and 12 weeks, none of implanted regions showed FA/ICGA leakage, some lesions had blockage due to hyperpigmentation; on OCT, representative areas showed preserved ellipsoid bands, with some RPE undulations. Lasered/implanted areas with a peripheral hyperpigmentation showed central outer retinal atrophy along with irregular RPE. Control implantation sites showed retinal atrophy and a variably thickened RPE band. Large-area RPE removal with laser disruption is feasible in healthy rabbits and appears to facilitate superior integration of RPE suspension grafts, compared to subretinal injection alone. Future work aims to correlate histology with in vivo imaging. This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually
Cardiac Subtype-Specific Modeling of Kv1.5 Ion Channel Deficiency Using Human Pluripotent Stem Cells
The ultrarapid delayed rectifier K+ current (IKur), mediated by Kv1.5 channels, constitutes a key component of the atrial action potential. Functional mutations in the underlying KCNA5 gene have been shown to cause hereditary forms of atrial fibrillation (AF). Here, we combine targeted genetic engineering with cardiac subtype-specific differentiation of human induced pluripotent stem cells (hiPSCs) to explore the role of Kv1.5 in atrial hiPSC-cardiomyocytes. CRISPR/Cas9-mediated mutagenesis of integration-free hiPSCs was employed to generate a functional KCNA5 knockout. This model as well as isogenic wild-type control hiPSCs could selectively be differentiated into ventricular or atrial cardiomyocytes at high efficiency, based on the specific manipulation of retinoic acid signaling. Investigation of electrophysiological properties in Kv1.5-deficient cardiomyocytes compared to isogenic controls revealed a strictly atrial-specific disease phentoype, characterized by cardiac subtype-specific field and action potential prolongation and loss of 4-aminopyridine sensitivity. Atrial Kv1.5-deficient cardiomyocytes did not show signs of arrhythmia under adrenergic stress conditions or upon inhibiting additional types of K+ current. Exposure of bulk cultures to carbachol lowered beating frequencies and promoted chaotic spontaneous beating in a stochastic manner. Low-frequency, electrical stimulation in single cells caused atrial and mutant-specific early afterdepolarizations, linking the loss of KCNA5 function to a putative trigger mechanism in familial AF. These results clarify for the first time the role of Kv1.5 in atrial hiPSC-cardiomyocytes and demonstrate the feasibility of cardiac subtype-specific disease modeling using engineered hiPSCs
Chromatin-Remodeling Components of the BAF Complex Facilitate Reprogramming
SummaryReprogramming of somatic cells achieved by combination of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has very low efficiency. To increase the reprogramming efficiency and better understand the process, we sought to identify factors that mediate reprogramming with higher efficiency. We established an assay to screen nuclear fractions from extracts of pluripotent mouse cells based on Oct4 reactivation. Using proteomics, we identified components of the ATP-dependent BAF chromatin-remodeling complex, which significantly increases reprogramming efficiency when used together with the four factors. The reprogrammed cells could transmit to the germline and exhibited pluripotency. Reprogramming remained highly efficient when c-Myc was not present but BAF components were overexpressed. BAF complex components mediate this effect by facilitating enhanced Oct4 binding to target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells
ExprEssence - Revealing the essence of differential experimental data in the context of an interaction/regulation net-work
<p>Abstract</p> <p>Background</p> <p>Experimentalists are overwhelmed by high-throughput data and there is an urgent need to condense information into simple hypotheses. For example, large amounts of microarray and deep sequencing data are becoming available, describing a variety of experimental conditions such as gene knockout and knockdown, the effect of interventions, and the differences between tissues and cell lines.</p> <p>Results</p> <p>To address this challenge, we developed a method, implemented as a Cytoscape plugin called <it>ExprEssence</it>. As input we take a network of interaction, stimulation and/or inhibition links between genes/proteins, and differential data, such as gene expression data, tracking an intervention or development in time. We condense the network, highlighting those links across which the largest changes can be observed. Highlighting is based on a simple formula inspired by the law of mass action. We can interactively modify the threshold for highlighting and instantaneously visualize results. We applied <it>ExprEssence </it>to three scenarios describing kidney podocyte biology, pluripotency and ageing: 1) We identify putative processes involved in podocyte (de-)differentiation and validate one prediction experimentally. 2) We predict and validate the expression level of a transcription factor involved in pluripotency. 3) Finally, we generate plausible hypotheses on the role of apoptosis, cell cycle deregulation and DNA repair in ageing data obtained from the hippocampus.</p> <p>Conclusion</p> <p>Reducing the size of gene/protein networks to the few links affected by large changes allows to screen for putative mechanistic relationships among the genes/proteins that are involved in adaptation to different experimental conditions, yielding important hypotheses, insights and suggestions for new experiments. We note that we do not focus on the identification of 'active subnetworks'. Instead we focus on the identification of single links (which may or may not form subnetworks), and these single links are much easier to validate experimentally than submodules. <it>ExprEssence </it>is available at <url>http://sourceforge.net/projects/expressence/</url>.</p
Epiblast Stem Cell Subpopulations Represent Mouse Embryos of Distinct Pregastrulation Stages
SummaryEmbryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast
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