Winding, P. & Berchtold, M.W. The chicken B cell line DT40: a novel tool for gene disruption experiments. J. Immunol. Meth. 249, 1-16
Institute of Molecular Biology, Øster Farimagsgade 2A, DK-1353, Copenhagen K, Denmark Journal of Immunological Methods
(Impact Factor: 1.82).
04/2001; 249(1-2):1-16. DOI: 10.1016/S0022-1759(00)00333-1
The use of the chicken DT40 B cell line is increasing in popularity due to the ease with which it can be manipulated genetically. It offers a targeted to random DNA integration ratio of more than 1:2, by far exceeding that of any mammalian cell line. The facility with which knockout cell lines can be generated, combined with a short generation time, makes the DT40 cell line attractive for phenotype analysis of single and multiple gene disruptions. Advantage has been taken of this to investigate such diverse fields as B cell antigen receptor (BCR) signaling, cell cycle regulation, gene conversion and apoptosis. In this review, we give a historical introduction and a practical guide to the use of the DT40 cell line, along with an overview of the main topics being researched using the DT40 cell line as a model system. These topics include B cell-specific subjects such as B cell signaling and Ig rearrangement, and subjects common to all cell types such as apoptosis, histones, mRNA modification, chromosomal maintenance and DNA repair. Attention is in each case brought to peculiarities of the DT40 cell line that are of relevance for the subject. Novel applications of the cell line, e.g., as a vector for gene targeting of human chromosomes, are also discussed in this review.
Available from: Antony Philip Jackson
- "It exhibits an extraordinarily high rate of homologous recombination, and this property greatly facilitates gene targeting . The DT40 cell-line has thus become established as an important tool for the study of a wide range of cell-biological phenomena . To understand the function of clathrin in more detail, we inactivated both endogenous copies of the clathrin gene in DT40 and replaced them with clathrin under the control of the Tet-Off inducible promoter. "
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ABSTRACT: We have previously deleted both endogenous copies of the clathrin heavy-chain gene in the chicken pre B-cell-line DT40 and replaced them with clathrin under the control of a tetracycline-regulatable promoter (Tet-Off). The originally derived cell-line DKO-S underwent apoptosis when clathrin expression was repressed. We have also described a cell-line DKO-R derived from DKO-S cells that was less sensitive to clathrin-depletion. Here we show that the restriction of transferrin uptake, resulting in iron deprivation, is responsible for the lethal consequence of clathrin-depletion. We further show that the DKO-R cells have up-regulated an anti-apoptotic survival pathway based on the chemokine SDF-1 and its receptor CXCR4. Our work clarifies several puzzling features of clathrin-depleted DT40 cells and reveals an example of how SDF-1/CXCR4 signalling can abrogate pro-apoptotic pathways and increase cell survival. We propose that the phenomenon described here has implications for the therapeutic approach to a variety of cancers.
Available from: onlinelibrary.wiley.com
- "But with the use of mouse embryonic stem (ES) cells and chicken DT40 cells, genomic DNA can be altered in its natural context by homologous recombination. Chicken B-lymphocyte-derived DT40 cells are known to have an exceptionally high rate of homologous recombination (Buerstedde & Takeda 1991; Winding & Berchtold 2001). Accordingly, it is possible not only to knockout a candidate regulatory region, but also to knockin the knock-out region. "
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ABSTRACT: The chicken Ig-β locus is organized by three cell-type-specific genes and two ubiquitously expressed genes. B-cell-specific DNase I hypersensitive sites (DHS) in that locus, including three present inside the flanking gene, were grouped into six regions and deleted. The deletions decreased Ig-β mRNA content to <0.1% of that of normal DT40 cells and converted epigenetic parameters such as histone modifications, CG methylation and DNase I hypersensitivity into inactive states. Knocked-in DHS regions into knock-out cells reactivated both transcription of the Ig-β gene and epigenetic parameters. Thus, the collaboration of the scattered regulatory regions was essential and sufficient not only for B-cell-specific transcription of the Ig-β gene, but also for the conversion of epigenetic parameters. On the basis of the knock-in studies, we determined the regions involved in the conversion and maintenance of the epigenetic parameters. These scattered regulatory regions were limited in vicinity such as in an intron of the gene, in the intergenic regions and in the introns of a flanking gene.
Available from: James Swenberg
- "The DT40 cell line is rather unique among higher eukaryotic cells in that it exhibits a high ratio of targeted to random integration of transfected DNA (Sonoda et al., 2001; Dhar et al., 2001). Due to the facility with which the DT40 line can be manipulated genetically, and the fact that DT40 mutants are observed to show a strong phenotypic resemblance to murine mutants with respect to genes involved in DNA recombination and repair (Sonoda et al., 2001), it has seen a steady growth in its use in genetic studies including immunoglobulin diversification, DNA repair, chromosome segregation, RNA metabolism and cell signaling (Winding and Berchtold, 2001; Yamazoe et al., 2004; Dhar et al., 2001). "
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ABSTRACT: Chemists continually synthesize myriad new chemicals (∼2,000/year), some of which make their way into the environment or otherwise pose possible threats to humans who potentially become exposed to the compounds. Regulators must determine whether these, along with the glut (∼80,000) of existing, chemicals are toxic and at what exposure levels. An important component of this determination is to ascertain the mode of action (MOA) of each compound as it relates to the pathway the compound uses to induce genotoxicity. Several assays have traditionally been used to reveal these effects to the genome: the Ames test, tests with yeast and mammalian cell lines, and animal studies. Previously, we described a new multi-well plate-based method which makes use of the DT40 isogenic cell line and its dozens of available mutants knocked out in DNA repair and cell cycle pathways and we now provide a detailed protocol of the further improvement of the assay. Although the DT40 line has existed for some time and has been used in numerous studies of DNA repair pathways, little use has been made of this valuable resource for toxicological investigations. Our method introduces the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide dye scheme determination of cell survival in a manner that greatly increases throughput and reduces cost while maintaining reasonable sensitivity. Although this new genotoxicity assay requires validation with many more mutagens before becoming an established, regulatory decision-making analysis tool, we believe that this method will be very advantageous if eventually added to the repertoire of those investigating MOAs of potentially genotoxic substances.
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