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Chapter 3
Generation of Mice from Embryonic Stem Cells using Tetraploid Embryos as Hosts
| 3.1 |
|
Introduction |
| 3.2 |
Experimental Design |
| 3.3 |
Materials |
| 3.3.1 |
Preparation of 4n embryos: mice and reagents |
| 3.3.2 |
Preparation of 4n embryos: equipment |
| 3.3.3 |
ES cells and culture conditions |
| 3.3.4 |
Micromanipulation system |
| 3.4 |
Methods |
| 3.4.1 |
Preparation of host embryos |
| 3.4.2 |
Electrofusion of 2-cell stage embryos |
| 3.4.3 |
Removal of the zona pellucida |
| 3.4.4 |
Generation of ES↔multiple 4n embryos by aggregation |
| 3.4.5 |
Generation of ES↔ 4n embryos by blastocyst injection |
| 3.4.6 |
Data acquisition |
| 3.5 |
Anticipated Results |
| 3.6 |
Discussion and Commentary |
| |
Troubleshooting Tetraploid Complimentation Assay |
| 3.7 |
Application Notes |
| 3.8 |
Summary Points |
|
Chapter 4
Bioreactor Design and Implementation
| 4.1 |
|
Introduction |
| 4.2 |
Experimental Methods and Materials |
| 4.2.1 |
General System Description |
| 4.2.2 |
Closed System |
| 4.2.3 |
Hollow Fiber Bioreactor |
| 4.2.4 |
CES Fluid Circuit |
| 4.2.5 |
Oxygenator Design |
| 4.2.6 |
Monitoring |
| 4.2.7 |
User Interface |
| 4.3 |
Data Acquisition and Interpretation |
| 4.4 |
Discussion and Commentary |
| 4.5 |
Troubleshooting Table |
| 4.6 |
Application Notes |
| 4.6.1 |
Therapeutic dose of MSC grown from a whole bone marrow sample |
| 4.6.2 |
Non-adherent cell culture: Kg1a cells grown in suspension |
| 4.7 |
Summary Points |
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