| Description |
vii, 205 pages : illustrations ; 24 cm |
| Contents |
Machine derived contents note: Preface -- Abbreviations -- 1 An Overview of Gene Targeting and Stem Cells -- Jim McWhir, Alison Thomson and David Russell -- 1.1 The murine embryonic stem cell system -- 1.2 Human ES cells -- 1.3 Gene targeting and ES cells in other species -- 1.4 Gene targeting -- 1.5 Nuclear transfer -- 1.6 Conclusion -- 2 Advances in Gene Targeting in Murine Embryonic Stem Cells -- Alan R Clarke -- 2.1 Introduction -- 2.2 Conventional gene-targeting approaches -- 2.3 Conditional alleles -- 2.4 Tracking mutations: coat color tagging -- 2.5 Enhancing gene-targeting efficiency -- 2.6 Targeted mutagenesis using triplex-forming oligonucleotides -- 2.7 Chromosome engineering -- 2.8 High-throughput gene targeting strategies -- 2.9 Summary -- 3 Nuclear Transfer with Murine ES Cells -- [author name to come] -- 3.1 Introduction -- 3.2 Factors affecting efficiency of nuclear transfer -- Protocol 3.1: Preparation of donor cells -- Protocol 3.2: Oocyte collection -- Protocol 3.3: Enucleation -- Protocol 3.4: Microinjection of ES cell nuclei into enucleated MII oocytes -- Protocol 3.5: Activation of reconstructed oocytes -- Protocol 3.6: Cloned embryo culture and transfer into surrogate mothers -- Protocol 3.7: Micromanipulation technique and preparation of tools -- Protocol 3.8: Alternative method for murine nuclear transfer -- 4 Gene Targeting in Sheep -- A. John Clark and Wei Cui -- 4.1 Introduction -- 4.2 Growth characteristics of primary somatic cells -- 4.3 Construction and transfection of gene-targeting vectors -- 4.4 Detecting targeted cells -- 4.5 NT with targeted sheep cells -- 4.6 The control of replicative senescence -- 4.7 Telomerase extension of proliferative lifespan -- 4.8 Nuclear transfer with telomerase immortalized cells -- 4.9 Concluding remarks -- Protocol 4.1: Isolation of sheep fetal fibroblasts -- Protocol 4.2: Electroporation and selection -- Protocol 4.3: Colony picking -- Protocol 4.4: Preparation of genomic DNA for PCR -- Protocol 4.5: Resuscitation of frozen cells from duplicate plate -- 5 RNA Interference -- Florence Wianny, Roberta J. Weber and Magdalena Zernicka-Goetz -- 5.1 Introduction -- 5.2 The mechanism -- 5.3 RNAi in mammalian somatic cells -- 5.4 RNAi in mammalian embryonic cells -- 5.5 Conclusion -- Protocol 5.1: Synthesis of dsRNA in vitro -- Protocol 5.2: Microinjection of dsRNA into mouse oocytes and embryos -- 6 Human Embryonal Carcinoma Cells: Surrogate Tools in the Study of Human Embryonic Stem Cells -- Jonathan S. Draper and Peter W. Andrews -- 6.1 Introduction -- 6.2 Embryonal carcinoma cells from the laboratory mouse -- 6.3 Human embryonal carcinoma cells -- 6.4 Human embryonic stem cells -- 6.5 Surface antigens as tools for analysis of EC cells -- Protocol 6.1: Flow cytochemistry and fluorescence-activated cell sorting (FACS) -- Protocol 6.2: In situ immunofluorescence staining for surface antigens -- Protocol 6.3: RNA in situ hybridization -- 7 Isolation of Human ES Cells -- L. Ahrlund-Richter and R Pederson -- 7.1 Introduction -- 7.2 Derivation of hES cells -- 7.3 Towards further optimized conditions -- Protocol 7.1: Immunosurgery: Separation of inner cell mass (ICM) from trophoectoderm cells (TE) -- 8 In-Vitro Culture and Differentiation of Human Embryonic Stem Cells -- Maya Schuldiner and Nissim Benvenisty -- 8.1 Introduction -- 8.2 Proliferation of human ES cells -- 8.3 Differentiation of human ES cells -- 8.4 Future prospects -- Protocol 8.1: Preparation of media -- Protocol 8.2: Passaging of human ES cells -- Protocol 8.3: Freezing of MEFs and human ES cells -- Protocol 8.4: Thawing of MEFs and human ES cells -- Protocol 8.5: Preparation of MEF cells -- Protocol 8.6: Preparation of MEF feeder layers -- Protocol 8.7: Formation of EBs -- 9 Transfection of Human Embryonic Stem Cells -- Helen Priddle -- 9.1 Introduction -- 9.2 Progress towards efficient transfection of hES cells -- 9.3 Future of gene targeting in human ES cells -- Protocol 9.1: Feeder-free culture of hES cells -- Protocol 9.2: Preparation of DNA for transfection -- Protocol 9.3: Assessment of transfection protocols by transient transfection -- Protocol 9.4: Optimization of antibiotic concentrations -- Protocol 9.5: Lipofection -- Protocol 9.6: Electroporation using gene pulser -- Protocol 9.7: Electroporation using multiporator -- Protocol 9.8: Expansion of hES colonies |
| Notes |
Formerly CIP. Uk |
| Bibliography |
Includes bibliographical references and index |
| Subject |
Gene targeting.
|
|
Embryonic stem cells.
|
|
Genetic Engineering -- methods.
|
|
Stem Cells -- cytology.
|
|
Gene Targeting -- methods.
|
|
Cell Differentiation -- genetics.
|
| Author |
Thomson, Alison.
|
|
McWhir, Jim.
|
| LC no. |
2004009191 |
| ISBN |
1859963609 paperback alkaline paper |
|
