Reviews and Protocols in DT40 Research Subcellular Biochemistry

Jean-Marie Buerstedde, Shunichi Takeda1402048955, 9781402048951, 9781402048968

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.

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Table of contents :
CONTENTS……Page 6
FOREWORD……Page 11
CHAPTER 1. DT40 GENE DISRUPTIONS: A HOW-TO FOR THE DESIGN AND THE CONSTRUCTION OF TARGETING VECTORS……Page 13
CHAPTER 2. IMMUNOGLOBULIN GENE CONVERSION OR HYPERMUTATION: THAT’S THE QUESTION……Page 22
CHAPTER 3. GENOME RESOURCES FOR THE DT40 COMMUNITY……Page 36
CHAPTER 4. CHROMOSOME ENGINEERING IN DT40 CELLS AND MAMMALIAN CENTROMERE FUNCTION……Page 49
CHAPTER 5. FUNCTION OF RECQ FAMILY HELICASE IN GENOME STABILITY……Page 59
CHAPTER 6. GENETIC ANALYSIS OF APOPTOTIC EXECUTION……Page 84
CHAPTER 7. THE DT40 SYSTEM AS A TOOL FOR ANALYZING KINETOCHORE ASSEMBLY……Page 100
CHAPTER 8. ANALYSING THE DNA DAMAGE AND REPLICATION CHECKPOINTS IN DT40 CELLS……Page 116
CHAPTER 9. USING DT40 TO STUDY CLATHRIN FUNCTION……Page 127
CHAPTER 10. GENETIC ANALYSIS OF B CELL SIGNALING……Page 152
CHAPTER 11. DT40 MUTANTS: A MODEL TO STUDY TRANSCRIPTIONAL REGULATION OF B CELL DEVELOPMENT AND FUNCTION……Page 195
CHAPTER 12. TRANSCRIPTION AND RNA PROCESSING FACTORS PLAY COMPLEX ROLES IN DT40 CELLS……Page 212
CHAPTER 13. PARTICIPATION OF HISTONES, HISTONE MODIFYING ENZYMES AND HISTONE CHAPERONES IN VERTEBRATE CELL FUNCTIONS……Page 229
CHAPTER 14. ANALYSIS OF GENE EXPRESSION, COPY NUMBER AND PALINDROME FORMATION WITH A DT40 ENRICHED CDNA MICROARRAY……Page 248
CHAPTER 15. CALCIUM SIGNALING, ION CHANNELS AND MORE……Page 260
CHAPTER 16. ANALYSIS OF DNA REPLICATION DAMAGE BYPASS AND ITS ROLE IN IMMUNOGLOBULIN REPERTOIRE DEVELOPMENT……Page 274
CHAPTER 17. THE FANCONI ANEMIA PATHWAY PROMOTES HOMOLOGOUS RECOMBINATION REPAIR IN DT40 CELL LINE……Page 298
CHAPTER 18. PHENOTYPIC ANALYSIS OF CELLULAR RESPONSES TO DNA DAMAGE……Page 315
CHAPTER 19. ATM, A PARADIGM FOR A STRESS-RESPONSIVE SIGNAL TRANSDUCER IN HIGHER VERTEBRATE CELLS……Page 328
METHOD 1. STABLE NON-TARGETED TRANSFECTION OF DT40……Page 341
METHOD 2. BASIC CELL CULTURE CONDITIONS……Page 345
METHOD 3. EXCISION OF FLOXED-DNA SEQUENCES BY TRANSIENT INDUCTION OF MER-CRE-MER……Page 347
METHOD 4. IMMUNOGLOBULIN GENE CONVERSION AND HYPERMUTATION ASSAY BY FACS……Page 350
METHOD 5. TARGET SCREENING BY PCR……Page 352
METHOD 6. MITOTIC INDEX DETERMINATION BY FLOW CYTOMETRY……Page 354
METHOD 7. CENTRIFUGAL ELUTRIATION AS A MEANS OF CELL CYCLE PHASE SEPARATION AND SYNCHRONISATION……Page 358
METHOD 8. PREPARATION OF GENOMIC DNA FOR MICROARRAY-BASED COMPARATIVE GENOME HYBRIDIZATION……Page 361
METHOD 9. ANALYSIS OF CELLULAR Mg[sup(2+)] IN DT40 CELLS……Page 370
METHOD 10. TRANSIENT TRANSFECTION OF DT40……Page 376
METHOD 11. RETROVIRAL TRANSDUCTION OF DT40……Page 380
METHOD 12. COLONY SURVIVAL ASSAY……Page 384
METHOD 13. SUBCLONING DT40 BY LIMITING DILUTION……Page 389
METHOD 14. SUBNUCLEAR IMMUNOFLUORESCENCE……Page 391
METHOD 15. SISTER CHROMATID EXCHANGE ASSAY……Page 395
METHOD 16. 2D CELL CYCLE ANALYSIS……Page 400
METHOD 17. PURIFICATION OF TAP-TAGGED PROTEINS BY TWO-STEP PULL DOWN FROM DT40 CELLS……Page 404
METHOD 18. SYNCHRONIZATION OF CELLS……Page 409
METHOD 19. TARGETED TRANSFECTION OF DT40 CELLS……Page 413
METHOD 20. LUCIFERASE REPORTER ASSAY……Page 416
METHOD 21. INDIRECT IMMUNOFLUORESCENCE MICROSCOPY……Page 419
METHOD 22. QUANTIFICATION OF RECEPTOR-MEDIATED ENDOCYTOSIS……Page 422
METHOD 23. MEASUREMENT OF DNA SYNTHESIS AND STRAND BREAKS USING ALKALINE SUCROSE DENSITY GRADIENT CENTRIFUGATION……Page 426
METHOD 24. ISOLATION OF NUCLEAR AND CYTOPLASMIC PROTEINS FROM DT40 CELL LINES……Page 430
COLOR PLATES……Page 432
C……Page 464
E……Page 465
I……Page 466
R……Page 467
Z……Page 468