Name: microinjection techniques for studying mitosis in the drosophila melanogaster syncytial embryo
Uploaded: 2009-09-15T13:00:00
Description: Watch this Scientific Journal Video about Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo at JoVE.com

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.

व्यंजनों: अंगूर का रस प्लेटें: <ul><li> 5.5g bacto अगर </li><li> 14.5 छ डेक्सट्रोज या ग्लूकोज </li><li> 7.15 छ sucrose </li><li> 45 मिलीलीटर अंगूर का रस ध्यान की (100% का रस). </li><li> 204.5 मिलीलीटर 2 0 एच </li><li> 625 μl 10N NaOH </li></ul> उबलते जब तक सभी स?...

<ol>
	<li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+dynamics+in+Drosophila.">Mitotic spindle dynamics in Drosophila.</a> Int Rev Cytol. 259, 139-172 (2007).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34, 1-15 (2002).</li><li>Morris, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+methods+for+analyzing+the+functions+of+kinesins+in+early+embryos.">Microinjection methods for analyzing the functions of kinesins in early embryos.</a> Methods Mol Biol. 164, 163-172 (2001).</li><li>Brust-Mascher, I., Sommi, P., Cheerambathur, D. K., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-5-dependent+poleward+flux+and+spindle+length+control+in+Drosophila+embryo+mitosis.">Kinesin-5-dependent poleward flux and spindle length control in Drosophila embryo mitosis.</a> Mol Biol Cell. 20, 1749-1762 (2009).</li><li>Brust-Mascher, I., Civelekoglu-Scholey, G., Kwon, M., Mogilner, A., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+for+anaphase+B:+role+of+three+mitotic+motors+in+a+switch+from+poleward+flux+to+spindle+elongation.">Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation.</a> Proc Natl Acad Sci U S A. 101, 15938-15943 (2004).</li><li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+flux+and+sliding+in+mitotic+spindles+of+Drosophila+embryos.">Microtubule flux and sliding in mitotic spindles of Drosophila embryos.</a> Mol Biol Cell. 13, 3967-3975 (2002).</li><li>Cheerambathur, D. K., Brust-Mascher, I., Civelekoglu-Scholey, G., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+partitioning+of+mitotic+kinesin-5+crosslinkers+between+microtubule-bound+and+freely+diffusing+states.">Dynamic partitioning of mitotic kinesin-5 crosslinkers between microtubule-bound and freely diffusing states.</a> J Cell Biol. 182, 421-428 (2008).</li><li>Cheerambathur, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+an+anaphase+B+switch:+predicted+role+for+a+microtubule+catastrophe+gradient.">Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient.</a> J Cell Biol. 177, 995-1004 (2007).</li><li>Kwon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromokinesin,+KLP3A,+dives+mitotic+spindle+pole+separation+during+prometaphase+and+anaphase+and+facilitates+chromatid+motility.">The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility.</a> Mol Biol Cell. 15, 219-233 (2004).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+coordination+of+three+mitotic+motors+in+Drosophila+embryos.">Functional coordination of three mitotic motors in Drosophila embryos.</a> Mol Biol Cell. 11, 241-253 (2000).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bipolar+kinesin,+KLP61F,+cross-links+microtubules+within+interpolar+microtubule+bundles+of+Drosophila+embryonic+mitotic+spindles.">The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.</a> J Cell Biol. 144, 125-138 (1999).</li><li>Sharp, D. J., Rogers, G. C., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+dynein+is+required+for+poleward+chromosome+movement+during+mitosis+in+Drosophila+embryos.">Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos.</a> Nat Cell Biol. 2, 922-930 (2000).</li><li>Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+microtubule-sliding+motors+position+mitotic+centrosomes+in+Drosophila+early+embryos.">Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.</a> Nat Cell Biol. 1, 51-54 (1999).</li><li>Waterman-Storer, C. M., Desai, A., Bulinski, J. C., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy,+a+method+to+visualize+the+dynamics+of+protein+assemblies+in+living+cells.">Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.</a> Curr Biol. 8, 1227-1230 (1998).</li></ol>

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?...

microinjection techniques for studying mitosis in the drosophila melanogaster syncytial embryo

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, &asymp;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.

Watch this Scientific Journal Video about Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo at JoVE.com

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

इस प्रोटोकॉल और उच्च संकल्प इमेजिंग microinjection के उपयोग का वर्णन<em&gt; ड्रोसोफिला मेलानोगास्टर</em&gt; Syncytial समसूत्री विभाजन अध्ययन भ्रूण.

Microinjection तकनीक में सूत्रीविभाजन अध्ययन के लिए ड्रोसोफिला मेलानोगास्टर Syncytial भ्रूण

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

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Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Dissection of Larval CNS in Drosophila Melanogaster

ड्रोसोफिला मेलानोगास्टर में लारवल सीएनएस के विच्छेदन

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Studying Aggression in Drosophila (fruit flies)

Studying Aggression in Drosophila fruit flies

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Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

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उच्च संकल्प प्रौढ़ ड्रोसोफिला मेलानोगास्टर में हरकत की वीडियो ट्रैकिंग

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Electrophysiological Recording in the Drosophila Embryo

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Electrophysiological रिकॉर्डिंग में ड्रोसोफिला भ्रूण

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

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निगरानी लारवल में हार्ट फंक्शन ड्रोसोफिला मेलानोगास्टर शारीरिक अध्ययन के लिए

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

पैच और आयन चैनल के अध्ययन के लिए दबाना छिड़काव तकनीक में व्यक्त Xenopus Oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Illustrating के गतिशील kinetochore प्रोटीन और रहने में क्रोमोजोम प्रस्ताव व्यवहार के द्वारा mitotic चेकप्वाइंट अध्ययन<em&gt; ड्रोसोफिला</em&gt; Syncytial भ्रूण

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Measurement of Lifespan in Drosophila melanogaster

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उम्र के मापन में<em&gt; ड्रोसोफिला मेलानोगास्टर</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

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की पोटा संबंधी मामले के माध्यम से इमेजिंग<em&gt; ड्रोसोफिला मेलानोगास्टर</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

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छोटे आरएनए प्रजाति के उत्तरी ब्लाट पता लगाने में बढ़ी<em&gt; ड्रोसोफिला मेलानोगास्टर</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...