Reviews and Protocols in DT40 Research Subcellular Biochemistry 1st Edition by Jean Marie Buerstedde, Shunichi Takeda 1402048955 9781402048951

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ISBN 10: 1402048955

ISBN 13: 9781402048951

Author: Jean-Marie Buerstedde, Shunichi Takeda

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.

Reviews and Protocols in DT40 Research Subcellular Biochemistry 1st Table of contents:

1. DT40 Gene Disruptions: A Guide to Designing and Constructing Targeting Vectors
2. Immunoglobulin Gene Conversion or Hypermutation
3. Genome Resources for the DT40 Community
4. Chromosome Engineering in DT40 Cells and Centromere Function
5. Function of RECQ Family Helicases in Genome Stability
6. Genetic Analysis of Apoptotic Execution
7. The DT40 System for Analyzing Kinetochore Assembly
8. DNA Damage and Replication Checkpoints in DT40 Cells
9. Using DT40 to Study Clathrin Function
10. Genetic Analysis of B Cell Signaling
11. Transcriptional Regulation of B Cell Development in DT40 Mutants
12. Transcription and RNA Processing Factors in DT40 Cells
13. Histones, Modifying Enzymes, and Chaperones in Vertebrate Cells
14. DT40 Enriched cDNA Microarray for Gene Expression Analysis
15. Calcium Signaling and Ion Channels in DT40 Cells
16. DNA Replication Damage Bypass and Immunoglobulin Repertoire Development
17. Fanconi Anemia Pathway in Homologous Recombination Repair
18. Phenotypic Analysis of Cellular Responses to DNA Damage
19. ATM as a Stress-Responsive Signal Transducer
20. Stable Non-Targeted Transfection in DT40
21. Basic Cell Culture Conditions
22. Floxed-DNA Excision Using MER-CRE-MER
23. Immunoglobulin Gene Conversion and Hypermutation Assay
24. PCR for Target Screening
25. Mitotic Index Determination via Flow Cytometry
26. Cell Cycle Synchronization Using Centrifugal Elutriation
27. Preparation of Genomic DNA for Microarray Analysis
28. Cellular Mg²⁺ Analysis in DT40 Cells
29. Transient Transfection of DT40 Cells
30. Retroviral Transduction of DT40
31. Colony Survival Assay
32. Subcloning DT40 by Limiting Dilution
33. Subnuclear Immunofluorescence
34. Sister Chromatid Exchange Assay
35. 2D Cell Cycle Analysis
36. Protein Purification Using Tap-Tagged Pull Down
37. Synchronization of Cells
38. Targeted Transfection of DT40 Cells
39. Luciferase Reporter Assay
40. Indirect Immunofluorescence Microscopy
41. Quantification of Receptor-Mediated Endocytosis
42. DNA Synthesis and Strand Break Measurement
43. Isolation of Nuclear and Cytoplasmic Proteins from DT40 Cells

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