Name: microinjection of mrna and morpholino antisense oligonucleotides in zebrafish embryos.
Uploaded: 2009-05-07T00:00:00
Description: Watch this Scientific Journal Video about Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos. at JoVE.com

Questo lavoro è stato supportato dal NIH e PKD fondazione a ZS. Tutti gli esperimenti sugli animali sono state condotte in base a Yale Animal Resources Center (Yarc) e cura degli animali istituzionali e Usa Committee (IACUC) le linee guida.

Parte 1: Preparazione di micropipette, e piatti da camera microiniezione <ol><li> Fabbricare micropipette da riscaldamento e tirando tubi in vetro borosilicato capillari (World Precision Instruments, Inc., 1B100-4) in un dispositivo estrattore micropipetta (Sutter Instruments Inc., Flaming / Brown P-97). Conservare in una capsula di Petri in cima alla piccola quantità di argilla o di nastro adesivo. </li><li> Versare 1,5% agarosio (American Bioanlytical, Inc., AB00972-00500) a 1x piatti medio E3 stampato con depressi...

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	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Nasevicius, A., Ekker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat Genet. 26, 216-220 (2000).</li><li>Draper, B., Morcos, P., Kimmel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+zebrafish+fgf8+pre-mRNA+splicing+with+morpholino+oligos:+a+quantifiable+method+for+gene+knockdown.">Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown.</a> Genesis. 30, 154-156 (2001).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+in+zebrafish+identifies+cilia+genes+as+a+principal+cause+of+cystic+kidney.">A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney.</a> Development. 131, 4085-4093 (2004).</li><li>Stuart, G., McMurray, J., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication,+integration+and+stable+germ-line+transmission+of+foreign+sequences+injected+into+early+zebrafish+embryos.">Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos.</a> Development. 103, 403-412 (1988).</li><li>Stuart, G., Vielkind, J., McMurray, J., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+lines+of+transgenic+zebrafish+exhibit+reproducible+patterns+of+transgene+expression.">Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression.</a> Development. 109, 577-584 (1990).</li><li>Culp, P., N&#252;sslein-Volhard, C., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+germ-line+transmission+of+plasmid+DNA+sequences+injected+into+fertilized+zebrafish+eggs.">High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs.</a> Proc Natl Acad Sci U S A. 88, 7953-7957 (1991).</li><li>Strehlow, D., Heinrich, G., Gilbert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fates+of+the+blastomeres+of+the+16-cell+zebrafish+embryo.">The fates of the blastomeres of the 16-cell zebrafish embryo.</a> Development. 120, 1791-1798 (1994).</li><li>Helde, K., Wilson, E., Cretekos, C., Grunwald, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+early+cells+to+the+fate+map+of+the+zebrafish+gastrula.">Contribution of early cells to the fate map of the zebrafish gastrula.</a> Science. 265, 517-520 (1994).</li><li>Lin, S., Long, W., Chen, J., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+germ-line+chimeras+in+zebrafish+by+cell+transplants+from+genetically+pigmented+to+albino+embryos.">Production of germ-line chimeras in zebrafish by cell transplants from genetically pigmented to albino embryos.</a> Proc Natl Acad Sci U S A. 89, 4519-4523 (1992).</li><li>Long, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATA-1+expression+pattern+can+be+recapitulated+in+living+transgenic+zebrafish+using+GFP+reporter+gene.">GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene.</a> Development. 124, 4105-4111 (1997).</li><li>Murphey, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+screening+in+the+zebrafish.">Small molecule screening in the zebrafish.</a> Methods. 39, 255-261 (2006).</li></ol>

Microiniezione in embrioni di zebrafish è una tecnica consolidata e robusta per esplorare il ruolo di un particolare gene nello sviluppo. Le applicazioni includono l&#39;iperespressione, misexpression e saggi knockdown del gene di interesse così come l&#39;analisi epistatico fra più geni. Microiniezione in zebrafish è stato ampiamente utilizzato per la generazione di animali transgenici, e mappatura destino delle cellule ai primi embrioni blastula 5,6,7,8,9. Inoltre, l&#39;applicazione di questa tecnica s...

Tutti gli altri materiali sono elencati nel protocollo.

microinjection of mrna and morpholino antisense oligonucleotides in zebrafish embryos.

Uno strumento essenziale per indagare il ruolo di un gene durante lo sviluppo è la capacità di eseguire knockdown gene, l&#39;iperespressione, e gli studi misexpression. In zebrafish (<em&gt; Danio rerio</em&gt;), La microiniezione di RNA, DNA, proteine, oligonucleotidi antisenso e di altre piccole molecole in sviluppo dell&#39;embrione fornisce ai ricercatori un test rapido e affidabile per esplorare la funzione del gene<em&gt; In vivo</em&gt;. In questo video-articolo, mostreremo come preparare e microinject<em&gt; In vitro</em&gt; Sintetizzato EGFP mRNA e una traslazione di blocco morfolino oligo contro<em&gt; PKD2</em&gt;, Un gene associato a malattia autosomica dominante del rene policistico (ADPKD), in 1 a cellule di embrioni fase zebrafish. Ci sarà quindi analizzare il successo del mRNA e microiniezioni morfolino verificando espressione GFP e analisi fenotipo. Vaste applicazioni di questa tecnica sono la generazione degli animali transgenici e la linea germinale chimere, cellule destino mappatura e lo screening del gene. Qui descriviamo un protocollo per la sovraespressione di EGFP e knockdown di<em&gt; PKD2</em&gt; Di mRNA e l&#39;iniezione oligonucleotide morfolino.

Watch this Scientific Journal Video about Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos. at JoVE.com

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

Microiniezione è un metodo consolidato ed efficace per l&#39;introduzione di sostanze estranee in embrioni di zebrafish fecondato. Qui, dimostriamo una tecnica robusta microiniezione per l&#39;esecuzione di sovraespressione di mRNA, e morfolino oligonucleotide studi gene knockdown in zebrafish.

Microiniezione di mRNA e oligonucleotidi antisenso Morpholino in embrioni di zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Research

JoVE Journal

Biology

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

Microiniezione di A. aegypti embrioni per ottenere zanzare transgeniche

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Ricerca

Biologia

Microinjection of Zebrafish Embryos to Analyze Gene Function

Microiniezione di embrioni di zebrafish per analizzare la funzione del gene

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Due fotoni assotomia e time-lapse confocale in embrioni di zebrafish vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Generating Chimeric Zebrafish Embryos by Transplantation

Generare embrioni di zebrafish Chimerico da trapianto

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Due-Photon-Based fotoattivazione in Live embrioni di zebrafish

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

Microiniezione di embrioni Medaka per essere utilizzato come organismo modello genetico

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

Analisi della cinetica di esportazione mRNA nucleare in cellule di mammifero da Microiniezione

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Glicani Imaging in embrioni di zebrafish con marcatura metabolica e Chimica Clicca Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Attivazione di apoptosi da Microiniezione citoplasmatici del Citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Metabolic Profile Analysis of Zebrafish Embryos

Analisi Profilo metabolico di embrioni di zebrafish

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...