Reviews and Protocols in DT40 Research

ISBN-13: 9781402048951 / Angielski / Twarda / 2006 / 492 str.

Buerstedde; Jean-Marie Buerstedde; Shunichi Takeda

Opis

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.

Spis treści

FOREWORD Chapter 1. DT40 GENE DISRUPTIONS: A HOW-TO FOR THE DESIGN AND THE CONSTRUCTION OF TARGETING VECTORS Hiroshi Arakawa and Jean-Marie Buerstedde CHAPTER 2. IMMUNOGLOBULIN GENE CONVERSION or HYPERMUTATION: That's the question Jean-Marie Buerstedde and Hiroshi Arakawa CHAPTER 3. Genome resources for the dt40 community Randolph B Caldwell and Andrzej M. Kierzek CHAPTER 4. Chromosome engineering in DT40 cells and mammalian centromere function William RA Brown1, Margaret CM Smith2, Felix Dafhnis-Calas, Sunir Malla and Zhengyao Xu. CHAPTER 5. FUNCTION OF RECQ FAMILY HELICASE IN GENOME STABILITY Masayuki Seki, Shusuke Tada and Takemi Enomoto chapter 6. Genetic analysis of apoptotic execution Sandrine Ruchaud, Kumiko Samejima, Damien Hudson, Scott H. Kaufmann and William C. Earnshaw CHAPTER 7. The DT40 system as a tool for analyzing kinetochore assembly Masahiro Okada, Tesuya Hori and Tatsuo Fukagawa CHAPTER 8. Analysing the DNA damage and Replication Checkpoints in DT40 cells Michael D Rainey, George Zachos, and David AF Gillespie CHAPTER 9. USING DT40 TO STUDY CLATHRIN FUNCTION Frank R. Wettey and Antony P. Jackson CHAPTER 10. GENETIC ANALYSIS OF B CELL SIGNALING Hisaaki Shinohara and Tomohiro Kurosaki CHAPTER 11. DT40 MUTANTS: A MODEL TO STUDY transcriptional regulation of B CELL DEVELOPMENT AND FUNCTION Jukka Alinikula, Olli Lassila and Kalle-Pekka Nera CHAPTER 12. TRANSCRIPTION AND RNA PROCESSING FACTORS PLAY COMPLEX TOLES IN DT40 CELLS Stephanie Bush and James L. Manley CHAPTER 13. PARTICIPATION OF HISTONES, HISTONE MODIFYING ENZYMES AND HISTONE CHAPERONES IN VERTEBRATE CELL FUNCTIONS Hidehiko Kikuchi, Hirak Kumar Barman, Masami Nakayama, Yasunari Takami and Tatsuo Nakayama CHAPTER 14. ANALYSIS OF GENE EXPRESSION, COPY NUMBER AND PALINDROME FORMATION WITH A DT40 ENRICHED cDNA Microarray Paul E. Neima1, Joan Burnside, Katrina Elsaesser, Harry Hwang, Bruce E. Clurman, Robert Kimmel and Jeff Delrow cHAPTER 15. Analysis of DNA replication damage bypass and its role in immunoglobulin repertoire development Julian E. Sale, Anna-Laura Ross and Laura J. Simpson CHAPTER 16. THE FANCONI ANEMIA PATHWAY PROMOTES HOMOLOGOUS RECOMBINATION REPAIR IN DT40 CELL LINE Julian E. Sale, Anna-Laura Ross and Laura J. Simpson CHAPTER 17. CALCIUM SIGNALING, ION CHANNELS AND MORE Anne-Laure Perraud, Carsten Schmitz, Andrew M. Scharenberg CHAPTER 18. Phenotypic analysis of Cellular responses to DNA damage Helfrid Hochegger and Shunichu Takeda CHAPTER 19. ATM, A PARADIGM FOR A STRESS-RESPONSIVE SIGNAL TRANSDUCER IN HIGHER VERTEBRATE CELLS Ken-ichi Yamamoto, Masahiko Kobayashi and Hiroko Shimizu METHOD 1. Stable transfection of DT40 Julian E. Sale METHOD 2. BASIC CELL CULTURE CONDITIONS Huseyin Saribasak and Hiroshi Arakawa METHOD 3. EXCISION OF FLOXED-DNA SEQUENCES BY TRANSIENT INDUCTION OF MER-CRE-MER Hiroshi Arakawa METHOD 4. IMMUNOGLOBULIN GENE CONVERSION AND HYPERMUTATION ASSAY BY FACS Hiroshi Arakawa METHOD 5. TARGET SCREENING BY PCR Hiroshi Arakawa METHOD 6. MITOTIC INDEX DETERMINATION BY FLOW CYTOMETRY David AF Gillespie and Mark Walker METHOD 7. Centrifugal elutriation as a means of cell cycle phase separation and synchronisation David AF Gillespie and Catarina Henriques METHOD 8. preparation of genomic dna for microarray-based comparative genome hybridization Robert Kimmel, Amalia Icreverzi, Paul Neiman METHOD 9. Analysis of cellular Mg2+ in DT40 cells Anne-Laure Perraud, Carsten Schmitz, Andrew M. Scharenberg METHOD 10. Transient transf