We thank Dr. C. Geoffrey Burns for fish stock. The authors are grateful to UNC Flow Cytometry Core Facility, UNC-CGIBD AAC core for resources enabling this project, and the ZAC facility for animal care. L.S. is supported by NIH T32 grant HL069768-13 (PI, Nobuyo Maeda). N.F. is supported by NSF Graduate Research Fellowship NSF-DGE-1144081. This study was supported by NIH P30DK034987 grant (to UNC Advanced Analytics Core), American Heart Association Scientist Development Grant 13SDG17060010 and Ellison Medical Foundation New Scholar Grant AG-NS-1064-13 (to Dr. Qian), and NIH R00 HL109079 grant (to Dr. Liu).

<ol>
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The authors have nothing to disclose.

השיטה המתוארת במסמך זה השתמש ביטוי של חלבון פלואורסצנטי תחת השליטה של ​​מקדם ספציפי תאים מהסוג להעשיר אוכלוסייה של ובתאים לב מעוברי דג זברה לשימוש במערכת ללכוד microfluidic בסיוע תא בודד להעריך ביטוי של תת-קבוצה של גני לב יחיד תאים. ובלבד יכולות עירור ופליטה לייזר FACS עול?...

רוב המחקרים הנוכחיים של ביולוגיה מולקולרית של תא מבוססים על ממוצעי אוכלוסייה. עם זאת, אירועים ביולוגיים חשובים עשויים להיות רעולי פנים על ידי ניתוחים מבוסס אוכלוסיה המסורתיים הללו, מאחר ואנשים קטין יכול לשחק תפקידים מרכזיים בתהליכים ביולוגיים ותוצאות המחלה. הבנת ביטוי גני אוכלוסיות הטרוגניות ברמת התא הבודדה יכולה (ויש לו) להוביל לתובנות ביולוגיים וקליניים רלוונטיים 1,2. דאגה ללימודי התפתחות עובריות, בתוך אוכלוסייה גדולה יותר של תאים, ובתאים הם בדרך כלל מיוצגים כראוי, מה שהופכים אותו מאתגר כדי לזהות שינויים עדינים ביטוי גנים שבסופו של דבר ליזום החלטות גורל תא 3. באופן דומה, סוג תא בודד יכול להיות ביטוי פרופילים שונים בתגובה במיקרו-סביבה של 4. לדוגמה, תאי אנדותל תושב אותו איבר או באיברים שונים (למשל., אבי העורקים או כליות) להפגין ההטרוגניות רבה, למרות שיתוף morp משותףתכונות hological ופונקציונליות 5. בנוסף, תאים סרטניים לאכלוס אותה הגידול יכולים להיות גם משתנים פרופילים או מוטציות מולקולריים ברמת התא הבודדה 6. במערכות מודל, transcriptomics יחידים תאים זיהה בהצלחת אוכלוסיות תאים חדשות, המאופיינת מצבי ביניים המתרחשים במהלך התמיינות תאים, וגילה תגובות הסלולר הפרש לגירויי 7,8,9. תובנות אלה היו רעולי פנים במחקרים מבוסס אוכלוסיה קונבנציונאלי. עוברי דג זברה הם מקור מתחת מנוצל מאוד של גזע, אב, ובידול תאים לחקר שאלות ההטרוגניות תא בודד ורגולציה מולקולרית של זהויות הסלולר במהלך פיתוח. פיתוח וקלות מניפולציה גנטית סטריאוטיפית שלהם מאוד, לשעבר vivo לגרום להם מערכת מודל מצוינת עבור גישה זו 10,11. באופן ספציפי, מגבלה משמעותית לפרשנות של תא בודד genנתוני דואר ביטוי הוא כי זיהוי אמין של מדינות תא ביניים רומן במהלך פיתוח דורש תזמון מאוד זהיר של אוסף רקמות 9. הדבר נחוץ כדי להבטיח כי ההטרוגניות בין תאים שנתפסו מייצג ההטרוגניות בתוך רקמה בנקודת זמן אחת ולא הטרוגניות בביטוי גנים שהציג התמיינות תאים תלוי גיל. בהשוואה לעכברים, פיתוח עובר דג זברה יכול להיות מסונכרן בדיוק על פני מספר גדול של עובר 12. בנוסף, עם גדלי מצמד גדולים, עוברי דג זברה יכולים לשמש מקור שופע של תאי גזע מולידם. פרוטוקול זה מתאר שיטה לבודד תאים מעוברים דג הזברה ללכוד תאים בודדים באמצעות מעגל מיקרופלואידיקה משולבת זמינים מסחרית (IFC) מערכת שבבים autoprep עבור ניתוח ביטוי גנים qRT-PCR. פרוטוקול זה יכול להיות להעברה מיד על כל מבחני ריבוב תפוקה גבוהה כולל כלרצף transcriptome המאפשר ניתוח מקיף יותר של ההטרוגניות הסלולר 13. הוא מציע גם מספר יתרונות מבחני ביטוי גנים מסורתיים. פרוטוקול בידוד תא הבודד מניב כדאיות גבוהה לאחר FACS, אשר מקטינה את חלקם של תאים נפגעים כלולי יישומים במורד זרם. באמצעות IFC, תאים שצולמו עשויים להיות שנצפו ישירות להעריך שיעורים ללכוד ולהעריך תא בריאות מורפולוגית. בנוסף, פרוטוקול זה הוא החלים רחב לקהילת מחקר דג הזברה, המחייב רק קו דג מהונדס שכותרתו וגישה לטכנולוגיות ללכוד תאי microfluidic. כהוכחה עקרונית, תאים בודדים נגזרו אבות לב הבודדים שנתפסו על שבב IFC, ולאחר מכן את השפע היחסי של סמני בידול לב נמדד על ידי qRT-PCR. ניתוח התבטאות גנים ברמת התא הבודדה מוכיח כי אבות לב לדור בכפיפה אחת עם differe שלהםצאצאי ntiating. התובנה שנצבר פרופיל תא בודד של אבות לב עשוי לשפוך אור על ההטרוגניות דפוסי ביטוי גנים בין ובתאים לב במהלך ההתפתחות חוליות, אשר עשויים להיות רעולי פנים בניתוחים מבוסס אוכלוסיה מסורתית.

<table><tbody><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>For removing the chorion from embryos</td></tr><tr><td>Microcentrifuge tube 2 ml</td><td>GeneMate</td><td>C-3261-1</td><td></td></tr><tr><td>40 um cell strainer</td><td>Biobasic</td><td>SP104151</td><td></td></tr><tr><td>35 mm culture dish</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>FACS tubes topped with 35 um cell strainer</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>P1000 and tips</td><td>Rainin</td><td>17005089</td><td></td></tr><tr><td>P20 and tips</td><td>Rainin</td><td>17005091</td><td></td></tr><tr><td>IFC chip manufacturer&#039;s protocol</td><td>Fluidigm</td><td>100-6117</td><td>Version 100-6117 E1 was used in representative experiment</td></tr><tr><td>Wide bore pastuer glass pippette</td><td>VWR</td><td>14673-010</td><td>For transferring embryos</td></tr><tr><td>Adult wild type zebrafish</td><td>N/A</td><td></td><td>We used AB line</td></tr><tr><td>Adult transgenic zebrafish</td><td>N/A</td><td></td><td>We used Tg(nkx2.5:ZsYellow)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for cell dissociation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Double distilled water</td><td>N/A</td><td></td><td></td></tr><tr><td>Instant Ocean Sea Salt</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>NaCl</td><td>FisherScientific</td><td>S271-3</td><td>make stock in water and use for de-yolking buffer</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P5405</td><td>make stock in water and use for de-yolking buffer</td></tr><tr><td>NaHCO3</td><td>Sigma Aldrich</td><td>S6014</td><td>make stock in water and use for de-yolking buffer</td></tr><tr><td>Leibovitz&#039;s L-15</td><td>Gibco</td><td>21083-027</td><td></td></tr><tr><td>FBS</td><td>FisherScientific</td><td>03-600-511</td><td>Heat inactivate; any brand of FBS should be fine</td></tr><tr><td>Cell Dissociation Reagent 1 -TrypLE</td><td>Life Technologies</td><td>12605-010</td><td>Store at room temperature.</td></tr><tr><td>Cell Dissociation Reagent 2 - FACSmax</td><td>Genlantis</td><td>T200100</td><td>Store -20; thaw on ice; bring to room temp before use</td></tr><tr><td>pronase (optional)</td><td>Sigma</td><td>P5147</td><td></td></tr><tr><td>L/D Dye - Sytox Blue</td><td>Life Technologies</td><td>S34857</td><td>Any live/dead stain suitable for flow cytometry will work</td></tr><tr><td>Trypan Blue</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for IFC plate use and qRT-PCR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gene-specific probes</td><td></td><td></td><td>Probes will vary by experiment</td></tr><tr><td>TaqMan Probe ef1a</td><td>Life Technologies</td><td>Dr03432748_m1</td><td></td></tr><tr><td>TaqMan Probe gata4</td><td>Life Technologies</td><td>Dr03443262_g1</td><td></td></tr><tr><td>TaqMan Probe nk2-5</td><td>Life Technologies</td><td>Dr03074126_m1</td><td></td></tr><tr><td>TaqMan Probe myl7</td><td>Life Technologies</td><td>Dr03105700_m1</td><td></td></tr><tr><td>TaqMan Probe vmhc</td><td>Life Technologies</td><td>Dr03431136_m1</td><td></td></tr><tr><td>TaqMan Probe isl1</td><td>Life Technologies</td><td>Dr03425734_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Life Technologies</td><td>4369016</td><td></td></tr><tr><td>Reagents listed in IFC manufacturer&#039;s protocol</td><td></td><td></td><td></td></tr><tr><td>C1 Reagent Kit</td><td>Fluidigm</td><td>100-5319</td><td>Reagents for loading cells onto IFC plate</td></tr><tr><td>Ambion Single Cell-to-CT&lt;sup&gt;TM&lt;/sup&gt;</td><td>Life Technologies</td><td>4458237</td><td>Reagents for reverse transcription and pre-amplification steps</td></tr><tr><td>Molecular Biology Quality Water</td><td>Corning</td><td>46-000CM</td><td></td></tr><tr><td>C1 IFC for PreAmp (5-10 um)</td><td>Fluidigm</td><td>100-5757</td><td>IFC plate for small cells</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C1 AutoPrep machine</td><td>Fluidigm</td><td>100-5477</td><td>For IFC plate use</td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Sigma Aldrich</td><td>Z359629</td><td>For counting cells and assessing cell size</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>For removing embryos from chorion</td></tr><tr><td>Tissue culture microscope</td><td></td><td></td><td>For assessing single cell digestion</td></tr><tr><td>FACS machine</td><td></td><td></td><td>For isolating cells of interest</td></tr></tbody></table>

isolation and characterization of single cells from zebrafish embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

כהוכחה עקרונית, ביטוי גנים הוערך לחקור דינמיקת בידול במהלך התפתחות של לב. בשנת דג זברה, אבות לב נובעים אוכלוסיית mesodermal של תאי נודדים אל mesoderm הצלחת לרוחב הקדמי שבו הם פתיל כדי ליצור את צינור הלב ליניארי. לפני היתוך, אבות לב מתחילים לבטא את nkx2.5 גור?...

Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

Isolation and Characterization of Single Cells from Zebrafish Embryos

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

בידוד ואפיון של תאים בודדים מעוברים דג הזברה

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

isolation-and-characterization-of-single-cells-from-zebrafish-embryos

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

14.4K Views.  University of North Carolina at Chapel Hill. This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells. 

Video: Isolation and Characterization of Single Cells from Zebrafish Embryos

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

סימון תאים הדמיה למוח האחורי של דג הזברה

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neuroscience

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

הכנת הדוגמא וניתוח של נתונים ביטוי גנים מבוססת RNASeq דג זברה

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Genetics

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

פרופיל תזמון שכפול ה-DNA באמצעות דג זברה כמערכת מודל In Vivo

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

חיים הדמיה של המוח על ידי עוברי דג הזברה מיקרוסקופיה confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biology

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

תרבות, תרביות תאים תאים ראשי דג זברה

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Preparation of Single-Somite Explants from Zebrafish Embryos

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

We present a protocol for isolating single somites from zebrafish embryos, the dynamics of which can be followed in culture for several hours by fluorescence time-lapse microscopy, thus providing a methodology to quantify tissue-scale shape changes at single-cell resolution.

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular ...

עיבוד תאי מעיים מזחלי דגי זברה לריצוף RNA חד-תאי

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

מחבר זרקור: בידוד וניתוח תאי מעיים של זחלי דגי זברה לחקר היבטים שעתוק של התפתחות מערכת העיכול 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

בידוד מעיים מזחלי דגי זברה לריצוף RNA חד-תאי

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

הר שלם ברזולוציה גבוהה<em&gt; באתרו</em&gt; הכלאה בתוך עוברי דג הזברה לחקר ביטוי גנים ותפקוד

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

בידוד ואפיון של תאים בודדים מעוברים דג הזברה

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בידוד מעיים מזחלי דגי זברה לריצוף RNA חד-תאי

בידוד מעיים מזחלי דגי זברה לריצוף RNA חד-תאי

הר שלם ברזולוציה גבוהה