ABSTRACT
OBJECTIVES: The aim of the present study was to assess the usefulness of insertion element IS1395 for differentiation of Mycobacterium xenopi, an increasingly common opportunistic human pathogen. METHODS: Fifty-two isolates obtained from 51 patients in Poland in 1996, 1997, and 1999, were analyzed by IS1395 restriction fragment length polymorphism (RFLP), and their susceptibilities to 11 anti-tuberculosis drugs were also determined. RESULTS: IS1395-associated banding patterns of the isolates were not highly polymorphic; the RFLP patterns displayed several bands in common. Nevertheless, 44 of the 52 isolates were clearly distinguishable from each other. Only eight strains (15.4%) occurred in four clusters of two identical clones, one of which comprised two isolates obtained from one patient with a 12-month interval. The remaining six patients with clustered strains showed no apparent epidemiologic links with the other patients from the same cluster, and they were most likely infected by the same environmental source. No noticeable difference in RFLP pattern or IS1395 copy number between drug-sensitive and drug-resistant strains was shown. A high proportion (84.6%) of strains resistant to at least one drug was found, and 7.7% were resistant to more than three drugs. CONCLUSIONS: The stability and utility of IS1395 for further detailed epidemiological investigations of M. xenopi was confirmed and extended.
Subject(s)
DNA Transposable Elements/genetics , DNA, Bacterial/genetics , Mycobacterium xenopi/genetics , Polymorphism, Restriction Fragment Length/genetics , Adult , Aged , Antitubercular Agents/pharmacology , Bacterial Typing Techniques , DNA Fingerprinting , Drug Resistance, Bacterial/genetics , Female , Humans , Male , Middle Aged , Molecular Epidemiology , Molecular Sequence Data , Mutagenesis, Insertional , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium xenopi/drug effects , Phylogeny , Poland , Polymerase Chain ReactionABSTRACT
Human stem and progenitor cells have recently become objects of intensive studies as an important target for gene therapy and regenerative medicine. Retroviral vectors are among the most effective tools for genetic modification of these cells. However, their transduction efficiency strongly depends on the choice of the ex vivo transduction system. The aim of this study was to elaborate a system for retroviral vector transduction of human CD34 positive cells isolated from cord blood. The retroviral vector pMINV EGFP was chosen for transduction of two human erythroblastoid cell lines: KG-1a (CD34 positive) and K562 (CD34 negative). For vector construction, three promoters and two retroviral vector packaging cell lines were used. To optimize the physicochemical conditions of the transduction process, different temperatures of supernatant harvesting, the influence of centrifugation and the presence of transduction enhancing agents were tested. The conditions elaborated with KG-1a cells were further applied for transduction of CD34 positive cells isolated from cord blood. The optimal efficiency of transduction of CD34 positive cells with pMINV EGFP retroviral vector (26% of EGFP positive cells), was obtained using infective vector with LTR retroviral promoter, produced by TE FLY GA MINV EGFP packaging cell line. The transduction was performed in the presence of serum, at 37 degrees C, with co-centrifugation of cells with viral supernatants and the use of transduction enhancing agents. This study confirmed that for gene transfer into CD34 positive cells, the detailed optimization of each element of the transduction process is of great importance.
