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  • 1
    Keywords: THERAPY ; ACTIVATION ; REPLICATION ; insertional mutagenesis ; HUMAN-IMMUNODEFICIENCY-VIRUS ; INTEGRATION ; REPOPULATING CELLS ; MARKING ; SCID-X1 ; SITE ANALYSIS
    Abstract: Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat-driven gamma-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus-long terminal repeat-driven gamma-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of linear amplification-mediated PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for linear amplification-mediated PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with gamma-retroviral long terminal repeat vectors.
    Type of Publication: Journal article published
    PubMed ID: 22989760
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  • 2
    ISSN: 1437-1588
    Keywords: Schlüsselwörter Tuberkulose ; Mykobakterien ; Mykobakteriendiagnostik ; Empfindlichkeitsprüfung ; Differenzierung ; Key words Tuberculosis ; Mycobacteria ; Primary isolation ; Susceptibility testing ; Species differentiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary At present conventional diagnosis of mycobacterial infections requires in case of posi- tive specimens about four weeks for culture followed by susceptibility testing. Negative results can be recorded after seven to eight weeks. Although some diagnostic proce- dures may be performed more rapidly applying new technologies like nucleic acid amplification techniques, at present this is still accompanied by a loss of sensitivity. The alarming global epidemiological situation of tuberculosis requires the development of new diagnostic technologies enabling rapid and accurate detection, differentiation, and susceptibility testing of mycobacteria.
    Notes: Zusammenfassung Die konventionelle Mykobakteriendiagnostik benötigt z.Zt. durchschnittlich vier Wochen bis zum Vorliegen einer positiven Tuberkulosekultur und nachfolgender Empfindlichkeitsprüfung. Bis zur Mitteilung eines negativen Kulturergebnisses vergehen sieben bis acht Wochen. Schnellere Resultate sind bei einzelnen diagnostischen Schritten durch neue Verfahren wie z.B. Nukleinsäure-Amplifikationstechniken zu erzielen, diese haben zur Zeit aber noch nicht die gewünschte hohe Sensitivität. Die weltweit beunruhigende epidemiologische Situation des Infektionsrisikos für Tuberkulose läßt keinen Zweifel aufkommen, daß die Weiterentwicklung von Methoden notwendig ist, die in möglichst kurzer Zeit gestatten, Mykobakterien mit hoher Sensitivität und Spezifität nachzuweisen, zu differenzieren und auf ihre Empfindlichkeit gegenüber Antibiotika zu testen.
    Type of Medium: Electronic Resource
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