Microinjection तकनीक में सूत्रीविभाजन अध्ययन के लिए ड्रोसोफिला मेलानोगास्टर Syncytial भ्रूण
Summary
इस प्रोटोकॉल और उच्च संकल्प इमेजिंग microinjection के उपयोग का वर्णन<em> ड्रोसोफिला मेलानोगास्टर</em> Syncytial समसूत्री विभाजन अध्ययन भ्रूण.
Abstract
This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) – tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2.
The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, ≈10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.
Protocol
Discussion
इस प्रोटोकॉल अपेक्षाकृत सरल है, तथापि, हर कदम अभ्यास की आवश्यकता को यकीन है कि भ्रूण को क्षतिग्रस्त नहीं हैं बनाने है. सावधानी से नियंत्रण प्रयोगों हमेशा करने के लिए सुनिश्चित करें कि विश्वसनीय परिणा?…
Acknowledgements
इस प्रोटोकॉल वर्तमान में हमारी प्रयोगशाला में प्रयोग किया जाता है और पिछले कुछ वर्षों में डीआरएस दाऊद तीव्र, Mijung Kwon करो, Patrizia Sommi, धन्य Cheerambathur सहित कई लोगों द्वारा परिष्कृत किया गया है. हम डॉ. विधेयक (UCSC) सुलेवान जो हेरफेर और जल्दी ड्रोसोफिला भ्रूण के microinjection जब इस प्रणाली में हमारे काम शुरू किया जा रहा था (Ref. 13) पर उत्कृष्ट सलाह के साथ हमें प्रदान धन्यवाद. हम Scholey प्रयोगशाला के सभी सदस्यों को धन्यवाद. ड्रोसोफिला में समसूत्री विभाजन पर हमारा काम NIH अनुदान GM55507 द्वारा समर्थित है.
References
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