מחקר זה נתמך על ידי תוכנית המחקר עירונית הלאומי לחקר הגנום האנושי, המכונים הלאומיים לבריאות.

חלק 1: הכנת הרקמה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את דג הזברה מבוגר עם שנאגרו MS-222 (אתיל 3-aminobenzoate מלח methanesulfonate, 0.03%). </li><li style=";text-align:right;direction:rtl"> לערוף את הדגים. חותכים את הלסת התחתונה, במקום הראש על כרית דיסקציה עם הצד הגחוני למעלה, לשתק את הרקמה עם סיכות חרקים. הסר את רקמות רכות הגחוני אל הגולגולת לפצח את החלק הגחוני של הקפסולה את הגולגולת עם פינצטה קנס תחת מיקרוסקופ לנתיחה. </li><li style=";text-align:right;direction:rtl"> תקן ראש דג paraformaldehyde (4%) ב -4 ° C במשך הלילה. </li><li style=";text-align:right;direction:rtl"> שטפו את ראש הדג קבוע PBS (פוספט בופר, 1X) במשך 3 פעמים, 5 דקות בכל פעם. </li></ol> חלק 2: דיסקציה הגולמי של האוזן הפנימית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את הראש על כרית דגים לנתיחה עם למעלה בצד הגחון. לשתק אותו עם סיכות חרקים. כל הצעדים הבאים מתבצעים תחת מיקרוסקופ לנתיחה. </li><li style=";text-align:right;direction:rtl"> עם פינצטה בסדר, להסיר את עצם הגולגולת מעל האוזן הפנימית ואז בזהירות לשלוף את רקמות האוזן הפנימית: saccules, lagenae ו utricles. שקיק ו lagena הם בחוזקה לצרף אחד את השני ובכך להוציא ביחד בעוד utricle (anterior את שקיק ו lagena), סביר מאוד להיות מנותקת מן האיברים הקצה השני במהלך לנתיחה. </li><li style=";text-align:right;direction:rtl"> שים את רקמות האוזן הפנימית של PBS (1X). אם לא לנתיחה קנס נוסף נדרש, יש לשטוף את הרקמה 2-3 פעמים עם PBS (1X) והוא יכול לשמש בניסויים אחרים, למשל cryosections. </li></ol> חלק 3: דיסקציה פיין epithelia של האוזן הפנימית החושית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דיסקציה הקנס יכול להתבצע ירידה של PBS (1X) בשקופית זכוכית נקי. </li><li style=";text-align:right;direction:rtl"> הסר את אבן האוזן utricular מ utricle עם שני זוגות של פינצטה בסדר, ואם תרצה, לקצץ את רקמת האפיתל ללא חושית ועל עצבוב מן epithelia עם פינצטה בסדר להב מחט. </li><li style=";text-align:right;direction:rtl"> הסר את אבן האוזן lagenar לפני הפרדת saccular ו lagenar epithelia בעוד מנסה לשמור על שקיק כולו שלם ככל האפשר. מניחים את הרקמה הירידה PBS עם למעלה בצד הגחון. השתמש בלהב המחט בזהירות לחתוך בין שקיק ו lagena. חתוך את הרקמה מופרז שאינו חושי אפיתל innvervation עם פינצטה בסדר הלהב מחט, אם תרצה בכך. </li></ol> חלק 4: מכתים פלורסנט של תאים שיער epithelia חושית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שוטפים את חושי epithelia עם PBT (1XPBS ואת טריטון 1% X-100) במשך 3 פעמים, 5 דקות בכל פעם. </li><li style=";text-align:right;direction:rtl"> דגירה epithelia עם fluophore שכותרתו phalloidin (למשל אלקסה 488 Phalloidin קמח, 1:1,000 דילול) למשך 30 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> שוטפים את חושי epithelia עם PBT במשך 3 פעמים, 10 דקות בכל פעם. </li><li style=";text-align:right;direction:rtl"> הר epithelia לשקופית עם הרכבה בינונית לבדוק את הרקמה תחת המיקרוסקופ מוכתם ניאון עם מסננים של הגל הנכון. </li><li style=";text-align:right;direction:rtl"> לאחר דיסקציה מכתים מוצלח, epithelia שלם עם תא חזקה מכתים שיער צרור ניתן לראות תחת מיקרוסקופ (איור 1). </li></ol><img src="/files/ftp_upload/1211/fig1.png" alt="איור 1" /> באיור 1. תאים שיער של האפיתל חושית saccular הוכתמו אלקסה 488 קמח Phalloidin (ירוק). דגימת הרקמה היה גזור ומוכתמת כאמור בעיתון. מספר תמונות צולמו בתנוחות שונות המדגם רעפים יחד עם Adobe Photoshop 7.0. בר סולם = 150μm.

בפרוטוקול זה, אנו תיאר את לנתיחה הגולמי קנס של epithelia האוזן הפנימית החושית של דג הזברה למבוגרים, אשר מאפשר לנו להכין דגימה לחקר תכונות הסלולר subcellular של epithelia החושי. על מנת לבצע דיסקציה מוצלח, הצעד הראשון מאוד, קיבעון, הוא מאוד חשוב. ו?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>MS222 (Ethyl 3-aminobenzoate methanesulfonate salt)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A5040</td>
<td>&nbsp;</td>
<tr>
<td>PBS (Phosphate-buffered Saline, 10X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>46-013-CM</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-6148</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 488 phalloidin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>A12379</td>
<td>&nbsp;</td>
<tr>
<td>VECTASHIELD Hard Set Mounting Medium with DAPI</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1500</td>
<td>&nbsp;</td>
<tr>
<td>Dissection microscope</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ1000</td>
<td>&nbsp;</td>
<tr>
<td>Insect pins</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>26000-40</td>
<td>&nbsp;</td>
<tr>
<td>Scissors</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-5880</td>
<td>&nbsp;</td>
<tr>
<td>Tweezers</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-5045</td>
<td>&nbsp;</td>
<tr>
<td>Needle blade</td>
<td>Tool</td>
<td>Sharpoint</td>
<td>78-6810</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Zeiss</td>
<td>Axiovert 200M</td>
<td>&nbsp;</td>
<tr>
<td>Dissection pad</td>
<td>Tool</td>
<td>eNasco</td>
<td>SB07840M</td>
<td>&nbsp;</td>
<tr>
<td>Zebrafish</td>
<td>Animal</td>
<td>various</td>
<td>n/a</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

gross and fine dissection of inner ear sensory epithelia in adult zebrafish (danio rerio)

נוירו epithelia באוזן הפנימית הם מבנים חיוני פונקציות השמיעה ושיווי המשקל. לכן, חשוב להבין את התכונות תאית ומולקולרית של האפיטל, אשר מורכב בעיקר שני סוגים של תאים: תאים שיער (HCS) ותאים תומכים (SCS). כאן אנו בוחרים ללמוד את epithelia האוזן הפנימית חושית דג הזברה מבוגר לא רק בגלל המבנים אפיתל הם שמור ביותר בכל חוליות למד, אלא גם משום דג הזברה מבוגר הוא מסוגל להתחדש HCS, יכולת כי היונקים לאבד זמן קצר לאחר הלידה. אנו משתמשים האוזן הפנימית של דג הזברה בוגר כמודל למערכת ללמוד את מנגנוני התחדשות פנימית HC אוזניים אצל בעלי החוליות מבוגר שיכול להיות מועיל עבור הטיפול הקליני של שמיעה / איזון גירעונות האדם כתוצאה של אובדן HC. כאן אנו מדגימים איך לעשות והניתוחים הגולמי קנס של epithelia האוזן הפנימית חושית דג הזברה מבוגר. דיסקציה ברוטו מסיר את הרקמות הסובבות את האוזן הפנימית מועיל להכנת סעיפים רקמות, אשר מאפשר לנו לבחון את המבנה המפורט של epithelia החושי. דיסקציה בסדר שמנקה את אי - חושית, רקמות אפיתל של האפיתל בנפרד ומאפשר לנו לבחון את ההטרוגניות של האפיתל כל בקלות כל הר דגימות אפיתל.

Watch this Scientific Journal Video about Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio at JoVE.com

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

האפיתל באוזן הפנימית החושית של דג הזברה מבוגר היא מערכת מודל טוב להבנת מנגנוני התחדשות התאים שיער חוליות מבוגר. פרוטוקול זה מדגים את לנתיחה קנס של האפיטל, שבאמצעותו אנו יכולים לקבל דגימות רקמה לחקר אירועי משובי ברמות הסלולר subcellular.

Dissection גרוס פיין epithelia של האוזן הפנימית חושית למבוגרים דג הזברה ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

gross-fine-dissection-inner-ear-sensory-epithelia-adult-zebrafish

Research

JoVE Journal

Biology

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

16.2K Views.  National Human Genome Research Institute. האפיתל באוזן הפנימית החושית של דג הזברה מבוגר היא מערכת מודל טוב להבנת מנגנוני התחדשות התאים שיער חוליות מבוגר. פרוטוקול זה מדגים את לנתיחה קנס של האפיטל, שבאמצעותו אנו יכולים לקבל דגימות רקמה לחקר אירועי משובי ברמות הסלולר subcellular.

Video: Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

Dissection ברוטו של קיבה של לובסטר, Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissection של איברים מן דג הזברה למבוגרים

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

משלוח ישיר של MIF Morpholinos Into Otocyst דג הזברה על ידי הזרקת electroporation משפיעה על פיתוח האוזן הפנימית

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Dissection של הכליה דג הזברה למבוגרים

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

הלב Dissection ב הזחל, דג הזברה לנוער ו למבוגרים, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

ב electroporation vivo של Morpholinos לתוך הרשתית דג הזברה למבוגרים

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

ב electroporation vivo של Morpholinos לתוך למבוגרים התחדשות זנב סנפיר דג הזברה

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

טיפול קבוע ותחזוקה של דג זברה (<em&gt; Danio rerio</em&gt;) מעבדה: מבוא

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

בדוגמנות Vivo של הגנום האנושי החולנית באמצעות Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging דג הזברה למבוגר

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...