The chicken B cell line DT40: a novel tool for gene disruption experiments
ABSTRACT 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.
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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.Genes to Cells 03/2011; 16(3):291-303. DOI:10.1111/j.1365-2443.2011.01486.x · 2.86 Impact Factor
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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.Environmental and Molecular Mutagenesis 03/2011; 52(2):153-60. DOI:10.1002/em.20595 · 2.55 Impact Factor
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ABSTRACT: Long interspersed elements (LINEs) are autonomous transposable elements that proliferate via retrotransposition, which involves reverse transcription of LINE RNAs. It is anticipated that LINE retrotransposition requires both LINE-encoded proteins and host-encoded proteins. However, identification of the host factors, their roles, and the steps at which they act on retrotransposition are poorly understood because of the lack of an appropriate genetic system to study LINE retrotransposition in a series of mutant hosts. To construct such a genetic system, we applied the retrotransposition-indicative cassette method to DT40 cells, a chicken cell line for which a variety of isogenic mutants have been established by gene targeting. Because DT40 cells are non-adherent, we utilized a selective soft agarose medium to allow the formation of colonies of cells that had undergone LINE retrotransposition. Colony formation was completely dependent on the activities of the LINE-encoded proteins and on the presence of the essential 3' region of the LINE RNA. Moreover, the selected colonies indeed carried retrotransposed LINE copies in their chromosomes, with integration features similar to those of genomic (native) LINE copies. This method thus allows the authentic selection of LINE-retrotransposed cells and the approximate recapitulation of retrotransposition events that occur in nature. Therefore, the DT40 cell system established here provides a powerful tool for the elucidation of LINE retrotransposition pathways, the host factors involved, and their roles.Gene 07/2007; 395(1-2):116-24. DOI:10.1016/j.gene.2007.02.017 · 2.08 Impact Factor