विकास तंत्रिका और खुली किताब की तैयारी में इंजेक्शन DiI phenotypes का आकलन ट्यूब में miRNA आधारित प्लास्मिड के ओवो electroporation
Summary
एक विधि है जिसके द्वारा न्यूरल ट्यूब में जीन की अभिव्यक्ति downregulated एक सेल प्रकार विशिष्ट, traceable तरीके से किया जा सकता है वर्णित है. हम का प्रदर्शन<em> ओवो</em> MicroRNA आधारित plasmids कि spatiotemporally नियंत्रित आरएनए हस्तक्षेप बटोर electroporation विकासशील न्यूरल ट्यूब में जोड़ संबंधी अक्षतंतु मार्गदर्शन की जांच करने के लिए इस्तेमाल किया जा सकता है.
Abstract
Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3′-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.
Protocol
Discussion
यह सरल, वेक्टर आधारित कृत्रिम miRNA अभिव्यक्ति रणनीति चिकन न्यूरल ट्यूब में पछाड़ना अंतर्जात जीन अभिव्यक्ति के लिए इस्तेमाल किया जा सकता है. इन कार्यात्मक उपकरण एकाधिक जीन मुंह बंद, अस्थायी नियंत्रण और ?…
Declarações
The authors have nothing to disclose.
Acknowledgements
ES की प्रयोगशाला में काम स्विस राष्ट्रीय विज्ञान फाउंडेशन द्वारा समर्थित है. हम फिल्माने के साथ सहायता के लिए डॉ. मारो Kunz धन्यवाद देना चाहूंगा.
Materials
| Name of reagent | Company | Catalogue number |
| 0.5 mm glass capillaries | World Precision Instruments | 1B120F-4 |
| Glass needle puller | Narishige | PC-10 |
| Electroporator | BTX | ECM 830 |
| Sylgard silicone elastomer | World Precision Instruments | SYLG184 |
| Tungsten wire, 0.075 mm | World Precision Instruments | TGW0325 |
| Insect pins, 0.20 mm | Fine Science Tools | 26002-20 |
| Insect pins, 0.10 mm | Fine Science Tools | 26002-10 |
| Spring scissors | Fine Science Tools | 15003-08 |
| Dumont #5 forceps | Fine Science Tools | 11252-20 |
| Dumont #55 forceps | Fine Science Tools | 11255-20 |
| Fast DiI | Molecular Probes | D-7756 |
| Fluorescent microscopes | Olympus | SZX12, BX51 |
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