המחברים רוצים להכיר את התמיכה על ידי &quot;Animalerie Centrale de Gif-sur-איווט&quot; לגידול בעלי חיים. עבודה זו נתמכה על ידי קרנות מANR (ANR-09-BLAN-0262-02 וANR-11-JSV2-002-01) למייקל שוברט, על ידי המענק של האיחוד האירופי FP6 &quot;Embryomics&quot; ועל ידי מענק ANR &quot;ANR- 10-BLAN-121,801 Dev-תהליך &quot;לז&#39;אן-פרנסואה ניקולא ונדין Peyriéras. João עמנואל Carvalho ממומן על ידי מענק דוקטורט FCT (SFRH / BD / 86878/2012). בקשות לווקטורים שתוארו כאן ניתן לפנות ישירות למחברים.

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The authors have nothing to disclose.

במאמר זה, אנו מציגים, בפעם הראשונה, פרוטוקול מפורט ושחזור להזרקה של B. ביציות lanceolatum, אשר, לאחר B. הדגלונים 13,14 ו- B. 15 belcheri, לכן מיני amphioxus שלישיים, שלטכניקה כזו שתוארה. חשוב לציין, הפרוטוקול מתואר כאן כולל גם את התיאור של מערכות וקטור המ?...

במהלך פיתוח, תא בודד מוליד כל אורגניזם בתהליך מורכב ביותר שיש בו שני חלוקות תא ותנועות. כדי להבין טוב יותר את העקרונות הביולוגיים העומדים בבסיס הדינמיקה של התנהגות תא, ביולוגים התפתחותיים החלו להשתמש בטכניקות ההדמיה vivo הקרינה מבוססת. תאים מסוימים של תאים, כגון קרום תא, יכולים גם להיות שכותרתו על ידי טיפולים עם צבעי ניאון, גישה הקשתה על ידי חוסר הייחוד ושל חדירת רקמת 1, או על ידי ההקדמה הספציפית לעובר של mRNAs אקסוגניים קידוד חלבוני ניאון 2. טכניקות שונות יכולות לשמש לאספקה ​​היעילה של תרכובות חיצוניות, כגון mRNAs. אלה כוללים, אך אינם מוגבלים ל, microinjection, electroporation, הפצצה עם microparticles, lipofection ותמרת 3,4. למרות שכל הגישות הללו יכולים לשמש כדי להציג את התרכובות אקסוגניים לפיתוח עובר, רק microinjection מאפשר היישום של כמויות מוגדרות מראש ומדויקות לכל תא 3. טכניקות Microinjection תוארו עבור כל מערכות המודל ההתפתחותי הגדולות 4 (לדוגמא, זבובי פירות, נמטודות, דג הזברה, צפרדעים, עכברים), כמו גם לכמה מודלים חלופיים 4, כוללים אלה המשמשים למחקרים השוואתיים שמטרתו להבין את האבולוציה של מנגנוני התפתחות (למשל, שושנות ים, תולעי תולעים טבעתיים, קיפודי ים, tunicates ascidian, amphioxus מיתרני הראש). Cephalochordates, אשר יחד עם tunicates ובעלי חוליות להקים מַעֲרָכָה chordate, במיוחד דגמים מתאימים היטב לחקר האבולוציה של chordates והגיוון של בעלי חוליות מאב קדמון חסר חוליות 5-8. שושלת מיתרני הראש התפצלה בשלב מוקדם מאוד במהלך האבולוציה chordate; וcephalochordates הקיים, אשר מחולקים לשלושה genera (Branchiostoma, Asymmetron וEpigonichthys), דומה לבעלי חוליות הן מבחינת האנטומיה כוללת ואדריכלות הגנום 5-8. של המינים על 30 של cephalochordates שתוארו עד כה, חמש זמינים למחקרים עובריים והתפתחותיים 6,9: lucayanum Asymmetron (אִזְמֵלוֹן בהאמה), מיתרני ראש הדגלונים (amphioxus פלורידה), lanceolatum מיתרני הראש (amphioxus האירופי), מיתרני הראש belcheri (amphioxus הסיני) ומיתרני הראש ג&#39;אפוניכאם (amphioxus היפני). מבוגרים הבשלים של שלושה ממינים אלו (lanceolatum ב &#39;, ב&#39; ו- B. belcheri ג&#39;אפוניכאם) יכולים להיגרם ללהשריץ על פי דרישה בעונת הרבייה 10,11. בנוסף, לפחות עבור B. lanceolatum, השרצה יעילה יכולה גם להיגרם במי ים מלאכותיים 12, ובכך הופכת את מיני מיתרני ראש מסוימים הזה נגיש לLaboratories שאין להם גישה למי ים טבעי. שילוב, בB. lanceolatum, של גישה נוחה ואמינה לעוברים עם שיטת אספקה ​​יעילה, כגון microinjection, עד כה טכניקת המשלוח בלבד שפותחה בamphioxus (בשני B. דגלונים וB. belcheri) 13-15, תאפשר הפיתוח של חבילת רומן של טכניקות מניפולטיביות, כולל tracing- שושלת וגישות המבוססת על התנהגות תא דינמי. פרוטוקול לmicroinjection יעיל של mRNAs לבטא חלבוני ניאון בB. עובר lanceolatum פותח ומכאן. יתר על כן, כדי לספק ערכת כלים בסיסיות להדמית חיה של B. עוברי lanceolatum, מערכות וקטור פותחו המאפשרות קרום הקשורים וביטוי גרעיני של חלבוני ניאון. למיקוד קרום, חלבון פלואורסצנטי ירוק משופר (eGFP) התמזג לתיבה האנושית HRAS CAAX ולוקליזציה גרעינית של mCherry וeGFP היהמתקבל על ידי היתוך לאקסון 2B היסטון דג הזברה (H2B) (איור 1, קובץ משלים 1). יתר על כן, במטרה לייעל את תרגום חלבון, הרצפים וקודונים קוזאק של המבנים שונו והותאמו לשימוש בB. lanceolatum. יחדיו, וקטורי שיטת ההזרקה וביטוי שהוצגו כאן ישמשו כבסיס לדור של גישות ניסיוניות החדשות לcephalochordates, בעיקר ניתוחים באמצעות מבוססת הקרינה האחרונה בטכניקות ההדמיה vivo.

<table border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td style="width:413px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Consumables</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Petri dishes</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>culture-treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Filtration unit (Stericup 1L)</td>
			<td style="width:156px;">Fisher</td>
			<td style="width:143px;">W21719</td>
			<td>0.22 micron filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Spin-X tubes</td>
			<td style="width:156px;">Costar</td>
			<td style="width:143px;">8160</td>
			<td>0.22 micron filtration tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Needle storage jar for 1.2 mm diameter capillaries</td>
			<td style="width:156px;">WPI</td>
			<td style="width:143px;">E212</td>
			<td style="width:413px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Pasteur Pipettes</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td>230 mm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Aspiration tube</td>
			<td style="width:156px;">Dutscher</td>
			<td style="width:143px;">75056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Capillaries for injection needles</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">BF 120-94-10</td>
			<td>Borosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reagents</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Low-melting agarose</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">A9414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol Red</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">114537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glycerol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Poly-L-Lysine hydrobromide</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P9155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">H2O Dnase, Rnase-free</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:143px;">10977-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">mMessage mMachine SP6 Transcription kit</td>
			<td style="width:156px;">Ambion</td>
			<td style="width:143px;">AM1340</td>
			<td>mRNA synthesis kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol pH8</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P4557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">24:1 chloroform:isoamylic alcohol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">C0549</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">5:1 phenol pH4.7:chloroform</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reef Crystal salts (200 kg)</td>
			<td style="width:156px;">Europrix&#160;</td>
			<td style="width:143px;"></td>
			<td>Commercial salts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Equipment</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Fluorescent dissecting scope with 200x magnification&#160;</td>
			<td style="width:156px;">Leica</td>
			<td style="width:143px;">MZ16F</td>
			<td>25x oculars, DSR and GFP2 filters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Micromanipulator</td>
			<td style="width:156px;">Marzhauzer</td>
			<td style="width:143px;">M-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Injector</td>
			<td style="width:156px;">Picospritzer</td>
			<td style="width:143px;">model II or III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Needle puller</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">P97</td>
			<td>heating-filament needle puller</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Fine forceps</td>
			<td>FINE SCIENCE TOOLS GMBH</td>
			<td>11252-30</td>
			<td>Dumont #5</td>
		</tr>
	</tbody>
</table>

expression of fluorescent proteins in branchiostoma lanceolatum by mrna injection into unfertilized oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

הפרוטוקול המפורט לעיל מספק את הבסיס לmicroinjection של B. ביציות lanceolatum ומכאן למבוא לפיתוח ב &#39; עוברי lanceolatum של mRNA קידוד חלבוני ניאון להדמית in vivo. למרות שהטכניקה היא בהחלט חזקה ואמינה, שיעור הזריקות מוצלחות תוך שימוש בפרוטוקול זה נשאר משתנה (טבלה ...

Watch this Scientific Journal Video about Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes at JoVE.com

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

אנו מדווחים כאן הביטוי חזק ויעיל של חלבוני ניאון לאחר הזרקת mRNA לביציות מופרה של lanceolatum מיתרני הראש. הפיתוח של טכניקת microinjection בchordate בסיס זה יסלול את הדרך למרחיקה לכת חידושים טכניים במערכת מודל מתפתח זה, לרבות בתחום ההדמיה vivo ומניפולציות גנטיות ספציפית.

ביטוי של חלבונים ניאון ב<em&gt; Lanceolatum מיתרני הראש</em&gt; על ידי הזרקת mRNA לביציות מופרה

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

expression-fluorescent-proteins-branchiostoma-lanceolatum-mrna

Research

JoVE Journal

Developmental Biology

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

10.9K Views.  Institut Pasteur.  אנו מדווחים כאן הביטוי חזק ויעיל של חלבוני ניאון לאחר הזרקת mRNA לביציות מופרה של lanceolatum מיתרני הראש. הפיתוח של טכניקת microinjection בchordate בסיס זה יסלול את הדרך למרחיקה לכת חידושים טכניים במערכת מודל מתפתח זה, לרבות בתחום ההדמיה vivo ו?...

Video: Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

ומניפולציה<em&gt; במבחנה</em&gt; ההבשלה של<em&gt; Laevis Xenopus</em&gt; ביציות, ואחריו הזרקת Intracytoplasmic זרע, לחקר התפתחות העוברית

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

העברת גנים לתוך האיברים השמיעתיים עוף ידי<em&gt; ב אובו</em&gt; מיקרו electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

אינדוקציה של התחדשות שריר שלד החריף ידי Cardiotoxin הזרקה

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

חיסונית-פלורסנט תיוג של Microtubules וחלבונים Centrosomal Vivo לשעבר רקמת המעי ו 3D במבחנה Organoids מעיים

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

ביטוי מהיר ויעיל של חלבונים מרובים בעוברי העופות באמצעות mRNA אלקטרופורציה

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

הומוגניים מתיכנות מראש של האדם פיברותקיעות על ידי ביטוי שנאכף של גורמים שעתוק

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls are composed of cellulose and non-cellulosic polysaccharides together with proteins, phenolic compounds and water. 90% of the cell wall is composed of&#160;polysaccharides (e.g., pectins) and arabinogalactan proteins (AGPs). The fluorescent immunolocalization of specific glycan epitopes in plant histological sections remains a key tool to uncover remodeling of wall polysaccharide networks, structure and components.
Here, we report an optimized fluorescent immunolocalization procedure to detect glycan epitopes from AGPs and pectins in plant tissues. Paraformaldehyde/glutaraldehyde fixation was used along with LR-White embedding of the plant samples, allowing for a better preservation of the tissue structure and composition. Thin sections of the embedded samples obtained with an ultra-microtome were used for immunolocalization with specific antibodies. This technique offers great resolution, high specificity, and the chance to detect multiple glycan epitopes in the same sample. This technique allows subcellular localization of glycans and detects their level of accumulation in the cell wall. It also permits the determination of spatio-temporal patterns of AGP and pectin distribution during developmental processes. The use of this tool may ultimately guide research directions and link glycans to specific functions in plants. Furthermore, the information obtained can complement biochemical and gene expression studies.

This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies.

אימונולוקליזציה פלואורסצנטית של חלבונים ערבינוגלקטניים ופקטינים בחומת התא של רקמות הצמח

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

כמות מינימלית של ויטריפיקציה של חתולים לא בוגרים

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

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 ...

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

הערכת שלמות מחסום הרכבת הציר בביציות עכבר

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...