This work was supported by NIH grants R21 HD068949-01 and RO1 GM065303.

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The authors do not have competing financial interests.

צעדים קריטיים זה קריטי לעבוד בתנאים של יעיל הפריה חוץ גופית. כדי להבטיח אספקה ​​טובה של ביציות בשלות (שלב 1), נקבות להגדיר עבור הזדווגות לא היה צריך להגדיר ב צלבי הזדווגות במשך 5 ימים לפחות, והיא אמורה להופיע הרה. במהלך ההפרעה של רבי?...

פרוטוקול זה מאפשר מניפולציה של ploidy עוברי דג הזברה, כגון בדור של diploids gynogenetic הומוזיגוטים מן haploids gynogenetic (איור 1) או לייצור tetraploids. זו מושגת על ידי גרימת עיכוב cytokinesis המתאים מחזור התא אחת בדיוק (איור 2 א, 2 ב). מהעיכוב של מחזור מפתח cytokinesis מושג על ידי טיפול עם הלם חום. הפרוטוקול הסטנדרטי של הלם תרמי (HS) כמו במקור שתיאר Streisinger ועמיתיו מעורבים דופק הטמפרטורה במהלך MPF תקופת 13-15, וכתוצאה מכך דוכן חלוקת התא חד מחזור במהלך מחזור התא הראשון 1. היעילות של פרוטוקול זה שופרה לאחרונה על ידי סריקת מחזורי התא מוקדם עם חלון זזה זמני של טיפול בהלם חום. סריקה זו מזוהית בשלב מאוחר יותר עבור הלם חום, עדיין בתוך מחזור התא הראשון (22-24 MPF), כי תוצאות שיעור גבוה יותר של embryos עם דוכן חלוקת התא חד מחזור, אשר במקרה זה משפיע על מחזור התא השני 2. התצפית כי מניפולציות ניסיון במהלך מחזור התא הראשון להפריע חלוקת תא במהלך מחזור התא השני ולגרום DNA תוכן שכפול גם דווחה מיני דגים אחרים 3,4. אנו מתייחסים פרוטוקול שונה זה כמו חום 2 הלם (HS2 - המונח &quot;2&quot; משקף כי דופק החום מתרחש בנקודת הזמן יאוחר שיטת HS הרגילה, וכי עיכוב המחזור לתאים הנגרמים HS2 מתרחש במהלך מחזור התא השני ). מחקרים אלה הראו כי בסיס מעצר cytokinesis לאחר הלם חום הוא העיכוב של שכפול צנטריול במהלך הדופק החום, אשר משפיע היווצרות ציר ואינדוקצית תלם מחזור התא הבא. תוצאות HS2 בתשואות של עיכוב מחזור התא מתקרבות 100%, ושיעורים עד שכפול ploidy עד 4 פעמים גבוהות יותר מאשר תקן HS 2. עוברים מטופלים שנינותחה הלם חום במהלך רכיבה על אופני תא blastomere מפגינים רבים השפעות מזיקות, הדבר המצביע על כך הלם החום משפיע מספר רב של תהליכים הנדרשים תא חלוק 2. מצד השני, אם הלם החום מוחל לפני תחילת רכיבת תא (פרק הזמן 0-30 MPF), זה משפיע כנראה עולה בקנה אחד עם הפרעה ספציפית עם שכפול צנטריול ואינו נראה להשפיע תהליכים בתא חיוניים אחרים 2 . מחקרים אלה הראו כי הזמן לפני התחלת חלוקת blastomere שנראה תקופת התפתחותית מקובלת באמצעות הלם חום ככלי לתמרן ploidy במיוחד באמצעות עיכוב צנטריול. הגורם הבסיסי של הסלקטיביות לכאורה הלם חום על שכפול צנטריול אינו ידוע, אך עשוי להיות קשור שפלה סלקטיבית של substructures centrosome נצפה תחת עומס חום בסוגי תאים מסוימים, כגון לויקוציטים 5. סנכרון זמני של דה העובריvelopment מושגת על ידי הפריה חוץ גופית (IVF). השימוש בתרומת זרע מגבר מטופל במהלך תוצאות הפריה בעוברים דיפלואידי שעם HS2 הנגרמת דוכן cytokinesis חד מחזור להפוך tetraploid. השימוש בתרומת זרע מגבר שטופלו UV, אשר נושאת crosslinks כי להשבית DNA, תוצאות בעוברים gynogenetic הפלואידים 6, שעם HSII הנגרמת דוכן cytokinesis חד מחזור להפוך 2 gynogenetic diploids. בגלל שכפול הגנום כולו וכתוצאה מכך, את diploids gynogenetic האחרון הם הומוזיגוטים בכל מוקד יחיד בגנום. לקבלת תמציתיות, אנו מתייחסים gynogenetic עוברים הפלואידים כמו &quot;haploids&quot;, ועוברים דיפלואידי gynogenetic הומוזיגוטים כמו &quot;diploids הומוזיגוטים&quot;. אם קיימא ופורה, ניתן להשתמש diploids הומוזיגוטים ליזום קווי סטרילי וקטלני חינם. homozygosity ישיר המושרה על ידי HS2 צריך גם לשלב בקלות ניתוח גנטי או מסכי גנטי, מאז diploids הומוזיגוטים מן הנקבות כי הם נשאים הטרוזיגוטיים של mutשיעורי תערוכת ations של homozygosity בבית גבוה וקבוע (50%) יחסים 2. הפרוטוקול הבא מתאר צעדים לביצוע HS2 ולגרום שכפול ploidy עם homozygosity מלא. לייצור tetraploid, פתרון הזרע צריך להיות מטופל. לייצור דיפלואידי הומוזיגוטים, הזרע צריך להיות מומת על ידי טיפול UV. בנוסף, כפי שמתואר הדיון, סמני פיגמנט גלויים יכולים לשמש גם כדי להקל על זיהוי של diploids הומוזיגוטים. תאומת דג זברה בעיקר במהלך השעות 3 הראשונות של הייזום של מחזור האור שלהם 7, ושניהם המבוגרים והביצים רגישות מקצבי יממת 8, כך להשגת התוצאות הטובות ביותר את תהליך ההזרעה המלאכותי אמור להתרחש בתוך פרק זמן זה.

<table><tbody><tr><td>Zebrafish mating boxes</td><td>Aqua Schwarz</td><td>SpawningBox1</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S3264</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;-7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>63138</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Tricaine</td><td>Western Chemical</td><td>Tricaine-D (MS 222)</td><td>FDA approved (ANADA 200-226)</td></tr><tr><td>Tris base</td><td>Sigma</td><td>77-86-1</td><td>to prepare 1 M Tris pH 9.0</td></tr><tr><td>HCl</td><td>Sigma</td><td>920-1</td><td>to prepare 1 M Tris pH 9.0</td></tr><tr><td>Fish net (fine mesh) (4-5 in)</td><td>PennPlax</td><td>(ThatFishThatPlace # 212370)</td><td>available in ThatFishThatPlace</td></tr><tr><td>Plastic spoon</td><td></td><td></td><td>available in most standard stores</td></tr><tr><td>Dissecting scissors</td><td>Fine Science Tools</td><td>14091-09</td><td></td></tr><tr><td>Dissecting forceps</td><td>Dumont</td><td>SS</td><td>available from Fine Science Tools</td></tr><tr><td>Dissecting stereoscope (with transmitted light source)</td><td>Nikon</td><td>SMZ645</td><td>or equivalent</td></tr><tr><td>Reflective light source (LED arms)</td><td>Fostec</td><td>KL1600 LED</td><td>or equivalent</td></tr><tr><td>Petri plates 10 cm diameter</td><td></td><td></td><td>any maker</td></tr><tr><td>Eppendorf tubes 1.5 ml</td><td></td><td></td><td>any maker</td></tr><tr><td>Ice bucket</td><td></td><td></td><td>any maker</td></tr><tr><td>Pipetteman P-1000</td><td></td><td></td><td>any maker</td></tr><tr><td>Pipette tips 1,000 &amp;micro;l</td><td></td><td></td><td>any maker</td></tr><tr><td>Narrow spatula</td><td>Fisher</td><td>14-374</td><td></td></tr><tr><td>Depression glass plate</td><td>Corning Inc</td><td>722085 (Fisher cat. No 13-748B)</td><td>available from Fisher Scientific</td></tr><tr><td>UV lamp</td><td>UVP</td><td>Model XX-15 (cat No. UVP18006201)</td><td>available from Fisher Scientific. Although not observed by us with this model, some UV sources have been observed to experience a decrease of intensity over time (if this is the case, see Modifications and Troubleshooting)</td></tr><tr><td>UV glasses</td><td></td><td></td><td>any maker</td></tr><tr><td>Paper towels</td><td></td><td></td><td>any maker</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark</td><td>06-666-11</td><td>available from Fisher Scientific</td></tr><tr><td>Timer stop watch</td><td></td><td></td><td>any maker</td></tr><tr><td>Wash bottle</td><td>Thermo Scientific</td><td>24020500</td><td>available from Fisher Scientific</td></tr><tr><td>Tea strainer</td><td></td><td></td><td>available in kitchen stores</td></tr><tr><td>beakers, 250 ml (2)</td><td>Corning Inc.</td><td>1000250</td><td>available from Fisher Scientific</td></tr><tr><td>water bath (2)</td><td></td><td></td><td>any maker, with accurary to 0.1 C (&lt;em&gt;e.g.&lt;/em&gt; Shel Lab H&lt;sub&gt;2&lt;/sub&gt;O Bath Series)</td></tr><tr><td>Hanks&amp;rsquo; Solution 1</td><td>see above</td><td>see above</td><td>8.0 g NaCl, 0.4 g KCl in 100 ml ddH&lt;sub&gt;2&lt;/sub&gt;O. Store at 4&amp;deg;C.</td></tr><tr><td>Hanks&amp;rsquo; Solution 2</td><td>see above</td><td>see above</td><td>0.358 g Anhydrous Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.6 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; in 100 ml ddH&lt;sub&gt;2&lt;/sub&gt;O. Store at 4 &amp;deg;C.</td></tr><tr><td>Hanks&amp;rsquo; Solution 4</td><td>see above</td><td>see above</td><td>0.72 g CaCl&lt;sub&gt;2&lt;/sub&gt; in 50 ml ddH&lt;sub&gt;2&lt;/sub&gt;O. Store at 4 &amp;deg;C.</td></tr><tr><td>Hank&#039;s Premix</td><td>see above</td><td>see above</td><td>add, in the following order: 10.0 ml Solution 1;&amp;nbsp; 1.0 ml Solution 2;&amp;nbsp; 1.0 ml Solution 4;&amp;nbsp; 86.0 ml ddH&lt;sub&gt;2&lt;/sub&gt;O;&amp;nbsp; 1.0 ml Solution 5. Store at 4 &amp;deg;C</td></tr><tr><td>Hanks&amp;rsquo; Solution 6</td><td>see above</td><td>see above</td><td>0.33 g NaHCO&lt;sub&gt;3&lt;/sub&gt; in 10 ml ddH&lt;sub&gt;2&lt;/sub&gt;O. Prepare fresh the morning of the IVF procedure.</td></tr><tr><td>Hank&#039;s Solution (final solution)</td><td>see above</td><td>see above</td><td>Combine 990 &amp;mu;l of Hank&amp;rsquo;s Premix and 10 &amp;mu;l of freshly made Solution 6 (NaHCO&lt;sub&gt;3&lt;/sub&gt; solution)</td></tr></tbody></table>

ploidy manipulation of zebrafish embryos with heat shock 2 treatment

מניפולציה של ploidy מאפשר טרנספורמציות שימושי, כגון diploids כדי tetraploids, או haploids כדי diploids. בשנתי ה rerio Danio דג הזברה, במיוחד בדור של diploids gynogenetic הומוזיגוטים שימושי בניתוח גנטי משום שהיא מאפשרת את הייצור הישיר של הומוזיגוטים מאם הטרוזיגוטיים יחידה. מאמר זה מתאר פרוטוקול שונה לשכפול ploidy מבוסס על הדופק חום במהלך מחזור התא הראשון, 2 הלם תרמי (HS2). באמצעות עיכוב של שכפול צנטריול, שיטה זו גורמת דוכן חלוקת התא מדויק במהלך מחזור התא השני. דוכן החלוקה חד מחזור המדויק, מצמיד את שכפול הדנ&quot;א מעושה, תוצאה הוא שכפול הגנום כולו. פרוטוקולים הקשורים בשיטה זו כוללים ביצת אוסף זרע, UV לטיפול זרע, הפריה חוץ גופייה ודופק חום לגרום לעיכוב חלוקת מחזור אחד תאים ושכפולים ploidy. גרסה שונה של פרוטוקול זה יכול להיות מיושמתploidy כדי לגרום לשינויים בעלי חיים אחרים.

למרות דוכן cytokinesis מחזור חד-תאי, שכפול ה- DNA מתרחש בדרך כלל עובר כאלה, וכתוצאה מכך שכפול של תוכן DNA של העובר (איור 1). פרוטוקול הלם חום Streisinger (HS הרגיל) כרוך דופק חום במהלך הפרית תקופת 13-15 דקות (MPF) ומשרת מעצר cytokinesis בעיקר במהלך חלוקת התא העוברית הראש...

Watch this Scientific Journal Video about Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment at JoVE.com

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

פרוטוקול שונה עבור מניפולציה ploidy משתמשת הלם החום להשפיע על דוכן אחד-מחזור ב cytokinesis בעובר המוקדם. פרוטוקול זה מודגם דג הזברה אבל עשוי לחול על מינים אחרים.

מניפולציה ploidy של עוברי דג הזברה עם חום 2 הלם טיפול

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

ploidy-manipulation-of-zebrafish-embryos-with-heat-shock-2-treatment

Research

JoVE Journal

Developmental Biology

10.0K Views.  University of Wisconsin.  פרוטוקול שונה עבור מניפולציה ploidy משתמשת הלם החום להשפיע על דוכן אחד-מחזור ב cytokinesis בעובר המוקדם. פרוטוקול זה מודגם דג הזברה אבל עשוי לחול על מינים אחרים. 

Video: Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

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 ...

Genetics

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

ביצוע דיפלואידי דג הזברה Gynogenetic על ידי לחץ מוקדמת

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Biology

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

מדמיין את המוח המתפתח בחיים דגים באמצעות הברקשת וזמן-הדמיה קונמיקוד

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

הפרדת תאים זקיקים וביצים בזקיקי השחלות של זברהפיש

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

ניטור מרחבי-טמפורלי מהיר ויעיל של מתילציה תקינה וחריגה של ציטוזין בתוך עוברים שלמים של דגי זברה

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

מערכת Tol2 של דג הזברה: גישת טרנסגנזה מודולרית וגמישה מבוססת שער

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

מתח ומות תא מפעיל באמצעות מיקרוסקופ Confocal הקונבנציונלי UV הליזר

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Neuroscience

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

ניתוח של סטרס חמצוני בעוברי דג הזברה

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

ייצור של הפלואידים עוברי דג הזברה על ידי<em&gt; במבחנה</em&gt; הפריה

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but protospacer adjacent motif (PAM) limitations are a major obstacle to creating accurate animal models of human genetic disorders caused by single-nucleotide variants (SNVs). Until now, some SpCas9 variants with broad PAM compatibility have shown efficiency in zebrafish. The application of the optimized SpRY-mediated adenine base editor (ABE), zSpRY-ABE8e, and synthetically modified gRNA in zebrafish has enabled efficient adenine-guanine base conversion without PAM restriction. Described here is a protocol for efficient adenine base editing without PAM limitation in zebrafish using zSpRY-ABE8e. By injecting a mixture of zSpRY-ABE8e mRNA and synthetically modified gRNA into zebrafish embryos, a zebrafish disease model was constructed with a precise mutation that simulated a pathogenic site of the TSR2 ribosome maturation factor (tsr2). This method provides a valuable tool for the establishment of accurate disease models for studying disease mechanisms and treatments.

This protocol describes how to perform efficient adenine base editing without PAM limitation to construct a precise zebrafish disease model using zSpRY-ABE8e.

עריכת בסיס יעילה ללא PAM עבור דגי זברה מידול של מחלות גנטיות אנושיות עם zSpRY-ABE8e

CRISPR/Cas9 technology has increased the value of zebrafish for modeling human genetic diseases, studying disease pathogenesis, and drug screening, but ...