चिकी midbrain डोपामिनर्जिक न्यूरॉन विकास में जीन समारोह के अध्ययन के लिए ओवो electroporation में
Summary
Midbrain डोपामिनर्जिक न्यूरॉन्स के विकास के दौरान समारोह और जीनों के विनियमन का आकलन करने के लिए, हम एक तरीका है कि शामिल है का वर्णन<em> ओवो में</emभ्रूण लड़की उदर मध्यमस्तिष्क डोपामिनर्जिक न्यूरॉन progenitors में प्लास्मिड डीएनए constructs के electroporation. इस तकनीक के लिए ब्याज की जीन की कुशल अभिव्यक्ति प्राप्त करने के लिए मध्यमस्तिष्क विकास और डोपामिनर्जिक न्यूरॉन भेदभाव के विभिन्न पहलुओं का अध्ययन करने के लिए इस्तेमाल किया जा सकता है.
Abstract
Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson’s disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick β-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.
Protocol
Discussion
भ्रूण लड़की मध्यमस्तिष्क के ओवो electroporation में एक कम लागत और ट्रांसजेनिक या पीटा जानवरों midbrain डोपामिनर्जिक न्यूरॉन के विकास में जीन कार्य के vivo अध्ययन में प्रदर्शन की पीढ़ी के लिए तेजी से विकल?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
हम प्रदान करने pCAG – mCherry निर्माण के लिए में डा. Takahiko Matsuda धन्यवाद. YCM Schweppe फाउंडेशन से कैरियर विकास पुरस्कार और व्हाइटहॉल फाउंडेशन से अनुदान द्वारा समर्थित है.
Materials
| Name of the reagent | Company | Catalogue Number | Comments |
| Fertilized chicken eggs | Charles River Laboratories | ||
| Egg incubator | GQF Manufacturing | 1502 Sportsman | |
| BTX Electroporator | BTX Harvard Apparatus | ECM830 | |
| Electrodes | BTX Harvard Apparatus | 45-0162 L-shaped genetrodes | For use with ECM830 electroporator |
| Platinum iridium wire | Alfa Aesar | #10056 | 0.5 mm diameter For making the 2 mm long electrodes |
| Glass capillary tube | World Precision Instrument | TW100F-4 | |
| Microloader pipette tip | Eppendorf | 5242 956.003 | |
| India ink | Staples | Filter 20% before use | |
| Microinjector | Parker Automation, Parker Hannifin Cooperation |
Picospritzer III | |
| Fluorescent dissection microscope | Leica | MZ16F | |
| Micropipette puller | Sutter Instrument | D-97 |
Riferimenti
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