Research is fast paced and advances in RNA interference have recently opened up new opportunities for genetic experiments in human cell lines. However, the possibility to easily modify the genome still remains a powerful tool to investigate the function of coding and regulatory sequences in the vertebrate genome. DT40 has never been a quick and easy road to fame. It would be unfair to blame this on DT40 as it has proven to be a reliable and robust companion with fast doubling time, easy clonability and a relatively stable karyotype. If this model system is going to flourish over the next 15 years, it will be thanks to ingenious and original researchers. They may feel as if they work outside the mainstream, but they can take heart by the fact that only the clever exploitation of diversity and conservation makes biological research both elegant and rewarding. It is with this in mind that the DT40 handbook has been conceived and written. This book provides an up to date overview of the different facets of research, and also intends to help newcomers get started and avoid looming pitfalls. The collection of protocols which have been kindly provided by a number of laboratories will be particularly useful in this regard.
Author(s): Hiroshi Arakawa, Jean-Marie Buerstedde (auth.), Jean-Marie Buerstedde, Shunichi Takeda (eds.)
Series: 40
Edition: 1
Publisher: Springer Netherlands
Year: 2006
Language: English
Pages: 477
Tags: Cell Biology; Cancer Research; Biochemistry, general; Life Sciences, general; Vertebrates
Front Matter....Pages i-xii
DT40 gene disruptions: a how-to for the design and the construction of targeting vectors....Pages 1-9
Immunoglobulin gene conversion or hypermutation: that's the question....Pages 11-24
Genome resources for the DT40 community....Pages 25-37
Chromosome engineering in DT40 cells and mammalian centromere function....Pages 39-48
Function of RECQ family helicase in genome stability....Pages 49-73
Genetic analysis of apoptotic execution....Pages 75-90
The DT40 system as a tool for analyzing kinetochore assembly....Pages 91-106
Analysing the DNA damage and replication checkpoints in DT40 cells....Pages 107-117
Using DT40 to study clathrin function....Pages 119-143
Genetic analysis of B cell signaling....Pages 145-187
DT40 mutants: a model to study transcriptional regulation of B cell development and function....Pages 189-205
Transcription and RNA processing factors play complex roles in DT40 cells....Pages 207-223
Participation of histones, histone modifying enzymes and histone chaperonesin vertebrate cell functions....Pages 225-243
Analysis of gene expression, copy number and palindrome formation with a DT40 enriched CDNA microarray....Pages 245-256
Calcium signaling, ion channels and more....Pages 257-270
Analysis of DNA replication damage bypass and its role in immunoglobulin repertoire development....Pages 271-294
The fanconi anemia pathway promotes homologous recombination repair in DT40 cell line....Pages 295-311
Phenotypic analysis of cellular responses to DNA damage....Pages 313-325
ATM, a paradigm for a stress-responsive signal transducer in higher vertebrate cells....Pages 327-339
Stable non-targeted transfection of DT40....Pages 341-344
Basic cell culture conditions....Pages 345-346
Excision of floxed-DNA sequences by transient induction of MER-CRE-MER....Pages 347-349
Immunoglobulin gene conversion and hypermutation assay by facs....Pages 351-352
Target screening by PCR....Pages 353-354
Mitotic index determination by flow cytometry....Pages 355-358
Centrifugal elutriation as a means of cell cycle phase separation and synchronisation....Pages 359-361
Preparation of genomic DNA for microarray-based comparative genome hybridization....Pages 363-371
Analysis of cellular Mg 2+ in DT40 cells....Pages 373-378
Transient transfection of DT40....Pages 379-382
Retroviral transduction of DT40....Pages 383-386
Colony survival assay....Pages 387-391
Subcloning DT40 by limiting dilution....Pages 393-394
Subnuclear immunofluorescence....Pages 395-398
Sister chromatid exchange assay....Pages 399-403
2D cell cycle analysis....Pages 405-408
Purification of tap-tagged proteins by two-step pull down from DT40 cells....Pages 409-413
Synchronization of cells....Pages 415-418
Targeted transfection of DT40 cells....Pages 419-421
Luciferase reporter assay....Pages 423-425
Indirect immunofluorescence microscopy....Pages 427-429
Quantification of receptor-mediated endocytosis....Pages 431-434
Measurement of DNA synthesis and strand breaks using alkaline sucrose density gradient centrifugation....Pages 435-438
Isolation of nuclear and cytoplasmic proteins from DT40 cell lines....Pages 439-440
Back Matter....Pages 441-477
