עבודה זו נתמכה על ידי הקרן הלאומית למדע IOS 0844948 וIOS 1024811

<ol>
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A., Bradham, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pantropic+retroviruses+as+a+transduction+tool+for+sea+urchin+embryos.">Pantropic retroviruses as a transduction tool for sea urchin embryos.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 5334-5339 (2012).</li></ol>

אין ניגודי אינטרסים שהוכרזו על ידי המחברים.

ישנם שני שלבים קריטיים שקשה עבור משתמשים מתחילים בטכניקה זו, אך הם חיוניים ליצירת הצלחת עוברי morphant. הוא הבחירה הראשונה ביציות בריאים שתבשלנה ולדשן כראוי. אחוז של התפתחות נורמלית בתרבות תלויה בעונה, על בריאותם של בעלי החיים, ואת מספר פעמים שביציות כבר נקצרו מאדם אחד. ב...

כוכב הים, Patiria miniata, (הידוע בכינויו כוכב העטלף) מסתמן כמערכת מודל מעניינת ותכליתית עבור מגוון רחב של 1 סלולארי 3, התפתחותיות 4,5, אבולוציוני 6 8, ומחקרים אקולוגיים 9- 11. פ למבוגרים miniata מופצים לאורך חוף האוקיינוס ​​השקט מסיטקה, אלסקה לאחה, 12 קליפורניה ונשמר בקלות באקווריום ימי. ביציות הן ימות השנה ברת השגה וכל נקבה יכולה לשפוך עשרות אלפי ביצים. ביציות הבשילו בקלות ומופרה חיצוני 13. העוברים וכתוצאה מכך הם שקופים ומאפשרים להתבוננות קלה; הם מפתחים באופן סינכרוני, ודורשים מים ים רק לפיתוח. הרכבה הגנום כולו וtranscriptomes מרובה זמינות גם עבור P. miniata <a href="http://www.echinobase.org/Echinobase/">(Echinobase.org).</a> יתרונות כגון להפוך אותם אידיאליים עבור מגוון רחב של מחקרומטרות הוראה. בשנים האחרונות, פ &#39; miniata הפך מערכת מודל לרשת רגולציה גן התפתחותי מנתחת 14- 16. מטרת מחקרים מסוג זה היא לזהות את כל המחמאה של גני רגולטורים ולקבוע את הרשת של יחסי הגומלין שלהם. חלק ניכר מהעבודה כרוכה בביטוי גנים מדאיג דרך כניסתה של oligonucleotides antisense או בmRNAs מבחנה מסונתזת. בנוסף, ניתוחי רגולציה cis משמשים כדי לאפיין את הפונקציה של DNA הרגולציה 15. ניתוחים אלה דורשים הקדמה של חומרים כימיים ההפרעות ו / או כתב הבונה DNA לעוברי. יתר על כן, כדי לאפיין את ההשפעות במורד הזרם של הפרעות אלה, יש assay עוברים רבים לשינויים בביטוי הגנים של מטרות פוטנציאליות. טכניקות לmicroinjection של מאות רבות של zygotes הן מרכזיות לעבודה זו. Echinoderms, כולל P. miniata </em&gt;, דורש חודשים רבים כדי להגיע לבגרות מינית. בגלל זה, זה בדרך כלל לא מעשי לפתח ולשמר קווים מהונדסים של בעלי חיים אלה לניסויים. לכן, גידול של מבוגרים מהונדסים לא יכול ביעילות ליצור הותאם עוברים. במקום זאת, צריכה להתרחש ההפרעות דה נובו באמצעות microinjection. Microinjection מציע הזדמנות לשנות עוברים עם ריאגנטים שאינם תא חדיר. הפרוטוקול הבא מתאר שיטה להציג את DNA, mRNA, קליעים נותבים תא, וoligonucleotides antisense morpholino למאות ביצים מופרות בשעה אחת 2-3 יושבת דרך microinjection. זה מייצר חומר מספיק עבור מגוון רחב של ניסויים במורד הזרם כולל, אך לא מוגבל ל, qPCR, הכלאה באתר, RNA-Seq, ומערבי סופג.

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			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
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			<td height="41" style="height:41px;width:216px;">1-Methyladenine</td>
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a method for microinjection of patiria minata zygotes

Echinoderms כבר זמן רב מערכת מודל מועדף למחקרים של רבייה והתפתחות, ולאחרונה לחקר ויסות הגנים והאבולוציה של תהליכים התפתחותיים. כוכב הים, Patiria miniata, הוא צובר שכיחות כמערכת מודל למחקרים מסוג שבוצעו באופן כמעט בלעדי בעבר בקיפודי הים, purpuratus Strongylocentrotus וvariegatus Lytechinus. יתרון של מערכות מודל אלה הוא הקלות של יצירת עוברים שונה שבגן מסוים הוא למעלה או downregulated, תיוג קבוצה של תאים, או החדרת גן כתב. שיטת microinjection בודדת מסוגלת לייצר מגוון רחב של עוברים שונה כגון. כאן, אנו מציגים שיטה לקבלת תאי רבייה מפ miniata, הפקת zygotes, והחדרת חומרים כימיים מדאיגים באמצעות microinjection. עוברי morphant בריאים מבודדים לאחר מכן למחקרים כמותיים ואיכותיים של geפונקצית ne. הזמינות של נתוני הגנום וtranscriptome לאורגניזם זה הגדילה את סוגי מחקרים שמבוצעים ואת הקלות של ביצועם.

המטרה של פרוטוקול זה היא להציג את חומרים כימיים לעוברי. אנו מדגימים את היעילות של הפרוטוקול על ידי הזרקה לבנות כתב DNA שמניע את הביטוי של חלבון פלואורסצנטי ירוק (GFP). עובר מוזרק להביע GFP בתיקונים משובטים (איור 4A-B) כDNA משלב במהלך המחשוף מוקדם. ריאגנטים רבים הנמצאי...

Watch this Scientific Journal Video about A Method for Microinjection of Patiria minata Zygotes at JoVE.com

A Method for Microinjection of Patiria minata Zygotes

שיטות המייצרות עוברי morphant חיוניות ללמוד מנגנוני התפתחות ורשתות רגולציה גן. כוכב הים Patiria miniata הוא מערכת מודל מתעורר ללימודים אלה. כאן אנו מציגים פרוטוקול לקבלת תאי רבייה, בהפקת תרבויות של עוברים, וmicroinjection המהיר של zygotes ממין זה.

שיטה לMicroinjection של<em&gt; Minata Patiria</em&gt; Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

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Biology

A Method for Microinjection of Patiria minata Zygotes

10.9K Views.  Carnegie Mellon University.  שיטות המייצרות עוברי morphant חיוניות ללמוד מנגנוני התפתחות ורשתות רגולציה גן. כוכב הים Patiria miniata הוא מערכת מודל מתעורר ללימודים אלה. כאן אנו מציגים פרוטוקול לקבלת תאי רבייה, בהפקת תרבויות של עוברים, וmicroinjection המהיר של zygotes ממין זה. 

Video: A Method for Microinjection of Patiria minata Zygotes

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

Microinjection עוברים א aegypti לקבל יתושים טראנסגנטי

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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

הדור של Stable טראנסגנטי C. elegans שימוש Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

שיטת לתרבות מוקדם של עוברים צ&#39;יק לשעבר vivo (תרבות חדש)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

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 both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Microinjection עוברים Medaka לשימוש כמודל אורגניזם גנטי

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

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

אנליזה של קינטיקה mRNA גרעיני ייצוא בתאי יונקים ידי Microinjection

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

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

טיהור הזרימה Cytometry מבוסס<em&gt; ס cerevisiae</em&gt; זיגוטות

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Microinjection מתא בודד לניתוח תקשורת נייד

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

התפוקה microinjections גבוה של zygotes קיפודי הים

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

בVivo Microinjection וElectroporation של אשך העכבר

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Microinjection דג הזברה למודל אי-ספיקת כליות חריפה

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...