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Expression vector system and a method for optimization and confirmation of DNA delivery and quantification of targeting frequency
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A FORSKOLIN AND VERAPAMIL SENSITIVE K+ CURRENT IN HUMAN TRACHEAL CELLS
No full textA voltage-dependent K+ current has been revealed in whole-cell recordings carried out on immortalized cells obtained from the human tracheal epithelium. At positive membrane potentials the current shows a time dependent inactivation which is accelerated by increasing the depolarizing step. Forskolin, a direct activator of adenylyl cyclase, and verapamil, a Ca2+ channel blocker, induce the K+ current to inactivate more rapidly. Control experiments show that the action of these two compounds is not mediated by cyclic AMP and Ca2+. The application of 1,9-dideoxyforskolin, an analogue which does not stimulate adenylate cyclase, inhibits the current in the same way as forskolin; on the contrary, the dibutyryl analogue of cyclic AMP is ineffective. Furthermore, eliminating extracellular Ca2+ does not affect K+ current kinetics. Tetraethylammonium is an effective blocker of this current with an IC50 of 0.3 mM
Genome medicine: gene therapy for the millennium, 30 September-3 October 2001, Rome, Italy
No full textThe recent surge of DNA sequence Information resulting from the efforts of agencies interested in deciphering the human genetic code has facilitated technological developments that have been critical in the identification of genes associated with numerous disease pathologies. In addition, these efforts have opened the door to the opportunity to develop novel genetic therapies to treat a broad range of inherited disorders, Through a joint effort by the University of Vermont, the University of Rome, Tor Vergata, University of Rome, La Sapienza, and the CSS Mendel Institute, Rome, an international meeting, 'Genome Medicine: Gene Therapy for the Millennium' was organized. This meeting provided a forum for the discussion of scientific and clinical advances stimulated by the explosion of sequence information generated by the Human Genome Project and the implications these advances have for gene therapy. The meeting had six sessions that focused on the functional evaluation of specific genes via biochemical analysis and through animal models, the development of novel therapeutic strategies involving gene targeting, artificial chromsomes, DNA delivery systems and non-embryonic stem cells, and on the ethical and social implications of these advances
VOLUME-SENSITIVE CHLORIDE CURRENTS IN 4 EPITHELIAL CELL LINES ARE NOT DIRECTLY CORRELATED TO THE EXPRESSION OF THE MDR-1 GENE
Full text linkIt has been shown recently that heterologous expression of human MDR-1 gene, which is responsible for multidrug resistance during cancer therapy, causes appearance of volume-sensitive Cl- currents, thus suggesting that the product of the MDR-1 gene (the P-glycoprotein) has a Cl- channel activity (Valverde, M. A., Diaz, M., Sepulveda, M. A., Gill, D. R., Hyde, S. C., and Higgins, C. F. (1992) Nature 355, 830-833). In the present work, we have tested four epithelial cell lines both for the expression of MDR-1 gene and for the presence of volume-sensitive Cl- currents. LoVo/H and LoVo/Dx cells derive from a human colon adenocarcinoma, the latter cell line being resistant to high concentrations of the antitumoral drug doxorubicin. 9HTEo- cells were obtained by transformation of human tracheal epithelium. The 9HTEo-/Dx cell line was established from these cells by selection in doxorubicin. As expected, higher levels of P-glycoprotein expression were detected in LoVo/Dx and 9HTEo-/Dx by means of reverse transcriptase polymerase chain reaction technique, indirect immunofluorescence, and Western immunoblot assays. In contrast with these data, the size of swelling-induced Cl- current was the same in the sensitive cell line and in its drug-resistant counterpart. Actually, the Cl- conductance of 9HTEo- and 9HTEo-/Dx was 4-fold higher than that of either LoVo/H or LoVo/Dx cells. This indicates that the amplitude of this conductance is not directly related to the expression of the MDR-1 gene
Targeted correction of a defective selectable marker gene in human epithelial cells by small DNA fragments
No full textA novel gene targeting strategy, small fragment homologous replacement (SFHR), has been used to correct specific genomic lesions in human epithelial cells. The frequency of targeting was estimated to be 1-10%. However, given the genomic target, the cystic fibrosis transmembrane conductance regulator (CFTR) gene, it is difficult to accurately quantify targeting frequency. As an alternative to targeting CFTR, targeted correction of a mutant selectable marker or reporter gene would be more amenable to accurate and rapid quantification of gene targeting efficiency. The present study evaluates the conditions that modulate SFHR-mediated correction of a defective Zeocin antibiotic resistance (Zeo(r)) gene that has been inactivated by a 4-bp insertion. The conditions include delivery systems, plasmid-to-fragment ratio, fragment length, and fragment strandedness (single- or double-stranded DNA). Targeting fragments comprise the wild-type Zeo(r) gene sequence and were either 410 (Zeo1) or 458 bp (Zeo3). Expression vectors containing the corrected Zeo(r) gene were isolated as episomal plasmids or were allowed to stably integrate into cultured human airway epithelial cells. Correction of the Zeo(r) gene was phenotypically defined as restoration of resistance to Zeocin in either bacteria or epithelial cell clones. Extrachromosomal gene correction was assayed using polymerase chain reaction amplification, restriction enzyme digestion, DNA sequencing, and Southern blot hybridization analysis of DNA from isolated prokaryotic and eukaryotic clones. Neither random sequence alteration in the target episomal gene nor random integration of the small fragments was detected. Targeted correction efficiencies of up to 4% were attained. These studies provide insight into parameters that can be modulated for the optimization of SFHR-mediated targeting
IONIC SELECTIVITY OF VOLUME-SENSITIVE CURRENTS IN HUMAN EPITHELIAL-CELLS
No full textThe ion selectivity of swelling-activated Cl- currents has been investigated in three different human epithelial cell lines, two derived from the airway epithelium (9HTEo- and CFNPE9o-) and one from a colon carcinoma (T84). The relative permeability of volume-sensitive currents with respect to Cl- is: I- (1.19) greater than NO3- (1.07) approximately Br-(1.05) greater than Cl-(1.0) greater than F-(0.5) approximately HCO3-(0.48) greater than isethionate(0.28) greater than aspartate (0.14) approximately gluconate(0.13) approximately SO4(2-)(0.12). This type of ion selectivity is similar to that described for depolarization-activated outwardly rectifying Cl- channels found in epithelial cells
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