We would like to thank Andrea Portbury and Davin Townley-Tilson for critical reading of the manuscript and the NIH (grant # R01HL061656) for funding support.

הערה: כל הניסויים אושרו על ידי הוועדה המוסדית הטיפול בבעלי חיים ושימוש באוניברסיטת צפון קרוליינה בצ&#39;אפל היל. .1 בלו מסתם אאורטלי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שימוש בטכניקת המתת חסד אושרה לשלב העוברי של עניין, להרדים עכבר בהריון עם גורים ביום הרצוי העוברי (E) של פיתוח. </li><li style=";text-align:right;direction:rtl"> השתמש 70% אתנול לעקר הבטן של הנשים בהריון. הרם את העור ושרירים של הבטן התחתונה ומן האיברים הפנימיים, ולנתח חלל הבטן התחתונה של פתוחה הנקבה כדי לאפשר גישה לקרן הרחם. </li><li style=";text-align:right;direction:rtl"> כדי לאחזר את קרן הרחם, להחזיק את צוואר הרחם עם מלקחיים וזנב לחתוך בזהירות למלקחיים. הרם את קרן הרחם, חיתוך כל רקמת חיבור שמחזיקה את הקרן במקום, ולהשלים את ההסרה על ידי חיתוך הצומת של קרן הרחם עם oviduct. </li><li style=";text-align:right;direction:rtl"> הנח את קרן הרחם גזור בcol צלחת פטרי המכילד 0.1 חיץ M טריס (pH 7.6) ולשטוף לפי צורך. לאחר מכן, חותך את קרן הרחם פתוח אורך חכם לחשוף את שקים העובריים. </li><li style=";text-align:right;direction:rtl"> כדי לחשוף את העובר, לחתוך לפתוח שק עוברי בצומת של השליה והעוברת ולחתוך את כלי הטבור לשחרר את העובר. מניחים את העובר גזור בצלחת פטרי שנייה עם חיץ M טריס קר 0.1. להחזיר את צלחת פטרי הראשונה עם עוברי undissected נותרים לקרח עד מוכן לעובר הבא. </li><li style=";text-align:right;direction:rtl"> כדי לשפר את קיר גישת חזה בתוך העובר, לערוף את העובר. אם גנוטיפ יש צורך, להסיר ולשמור חתיכת רקמה נוספת בצינור Eppendorf הניח על קרח. </li><li style=";text-align:right;direction:rtl"> מניחים את העובר על הגב שלה, וחתך את קיר החזה בצורה אנכית בצד של כלוב הצלעות, ליד forelimb, ואופקית מעל הסרעפת לדמיין את הלב. לאחר מכן, לפתוח את בית החזה כדי לחשוף את הלב. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים להחזיק גבוה לאורך כלי הגדול, להרים את הלב באמצעות מלקחיים ולחתוך את כלי להלן.n, לחתוך מעל המלקחיים כדי לשחרר את הלב. אם כלי ריאתי להישאר נימול, הריאות ניתן להסיר עם הלב, ויש להסיר לפני שתמשיך. </li><li style=";text-align:right;direction:rtl"> לחתוך ולהסיר את עורק הריאה מעל לרמה של השסתום; בשלבים העובריים שתוארו במסמך זה, עורק הריאה הוא מעט אטום, אשר מסייע באפליה שלה מאזור שסתום. לחתוך ממש מתחת לשסתום ריאתי, הימנעות שריר הלב של חדר trabeculated ולאסוף כפי שמתואר בשלב 1.10 אם אזור זה גם הוא עניין. כאן ובשלב הבא, להשתמש בפעולת נימים לצייר חיץ טריס עודף מהאזור של עניין לפני האיסוף במאגר תמוגה. </li><li style=";text-align:right;direction:rtl"> מוציא בזהירות את דיסטלי אב העורקים לשסתום אב העורקים, ממש מעל הרמה של השסתום; כמו עורק הריאה, אב העורקים נשארים אטומים מעט בשלבים העובריים אלה, בסיוע בהסרתה. לחתוך ממש מתחת לשסתום אב העורקים, שוב הימנעות שריר הלב של חדר trabeculated, ולהעביר אתשסתום באמצעות מלקחיים לצינור Eppendorf עם חיץ תמוגה 2 μl (שמתואר בטבלת חומרים) המכיל מעכבי פרוטאז וphosphatase על קרח. </li><li style=";text-align:right;direction:rtl"> חזור על שלבים 1.6-1.10 עד שסתומים כבר נכרת מכל העוברים, איסוף כל שסתום בצינור Eppendorf נפרד. אם יש לי כל העוברים גנוטיפ זהה, כל השסתומים עשויים להיות שנאספו בצינור Eppendorf יחיד. </li><li style=";text-align:right;direction:rtl"> אם גנוטיפ הוא שיש לבצע כדי לקבוע אילו שסתומי לחבור יחדיו, להציב מייד דגימות במאגר תמוגה ב -80 מעלות צלזיוס עד לשימוש נוסף. אז יכולות להיות ונקוו דגימות לאחר genotyping (צעד 2.1.1). </li></ol> הפקת .2 חלבון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להפשיר רקמות שנאספו על קרח ו צנטריפוגות ב XG 13,100 עבור 1 דקות כדי לאסוף את הרקמות במאגר תמוגה בחלק התחתון של הצינור. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אם עוברים עברו מיפוי, להשתמש פיפטה לאסוף בעדינות ובמאגר תמוגה בריכה המכיל את אזורי שסתום מעוברים עם גנוטיפים דומים. כדי להבטיח ב חזקו, איגום 3-4 אזורים שסתום מעוברי E13.5 לדגימה מומלצת. </li><li style=";text-align:right;direction:rtl"> אם כל העוברים היו של אותו גנוטיפ וכל אזורי שסתום נאספו יחד בשלב 1.10, lyse המדגם כולו, כפי שמתואר בשלב 2.2. </li></ol></li><li style=";text-align:right;direction:rtl"> בדוק את עוצמת הקול של המדגם נקווה כתוצאה ולהוסיף למאגר תמוגה להיקף כולל של 40 μl; לאחר מכן, לשבש וhomogenize דגימות באמצעות חרוזים נירוסטה 5 מ&quot;מ בצינורות אפנדורף. פועל lyser ל2-4 דק &#39;ב 50 הרץ, לפי הוראות היצרן. צינורות ספין בקצרה, (~ 13,100 XG 1 דקות), והעברת דגימות לצינורות חדשים, והותירו אחריו את הפסולת בלתי מסיס. </li><li style=";text-align:right;direction:rtl"> הכן דגימות נפרדות לSDS-PAGE והעברה לניתוח כתם מערבי שלאחר מכן, בעקבות פרוטוקול כגון אסלאמי et al 9. סופג לחלבון בקרת טעינה הוא חיוני לכימות חלבון. </li></ol>

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The authors have nothing to disclose.

היכולת לכמת רמות חלבון בעכבר עוברי מוקדם וחומוס אזורי שסתום לב מספקת כלי נוסף להבנת אירועי איתות התאיות הקריטיים להתפתחות שסתום. הפרוטוקול שלנו שתואר במסמך זה אינו שונה בהרבה מהנהלים בידוד חלבון סטנדרטיים. עם זאת, על ידי שינוי כמה שלבים עיקריים, שהשגנו בהצלחה חלבוני...

יכולת לזהות ולכמת רמות ביטוי חלבון היא טכניקה סטנדרטית לבהמה וניסויים מבוססי תאים. עם זאת, למרות ארוך שנתי עניין בפיתוח שסתום לב עוברי מוקדם, הערכת ביטוי חלבון ברקמה הספציפית הזה במהלך ההתפתחות כיום מוגבלת לאימונוהיסטוכימיה בשני האפרוח ו1,2 עכבר. חלק מהקושי של כימות ביטוי חלבון בשסתומי הפיתוח של רוב האורגניזמים מודל (למשל, אפרוח ועכבר) הוא בגודל הקטן של השסתומים, אשר מגביל את כמות החלבון שניתן להשיג. לפיכך, לניתוחים כמותיים, חוקרים בדרך כלל מסתמכים על מיצוי RNA והגברה לPCR כמו שלאחר מכן או ניתוח microarray 2-5. עם זאת, רמות ביטוי RNA והחלבון אינן מלא גומלות 6, כל כך מתמקדות בביטוי RNA אינו יכולות לספק חשבון קפדני של שינויים הרבים המתרחשים בכל איתות שניתנהמסלול בזמנים שונים במהלך התפתחות. בהתבסס על מגבלה זו במתודולוגיה זמינה כרגע, המטרה של הליך זה הייתה לפתח פרוטוקול למהימן קבלת כמות מספקת של חלבון מאזורי שסתום לב העכבר עוברי מתפתחים לניתוח כמותי של שינויים המתרחשים במסלולי איתות שונים שחשובים ב ההבשלה של רקמה זו. שסתומים עובריים כבר הם גזורים בדרך כלל מעכברים לבידוד RNA וניתוח ביטוי גנים לאחר 2-5. עם זאת, מחקרים אלו היו מוגבלים לשימוש בביטוי גנים כקריאה מתוך איתות הפעלת מסלול, שאינו מאפשר זיהוי של לאתר שינויי חלבון לאחר translational שעשוי להשפיע על איתות במורד הזרם. תוך שימוש בטכניקות בידוד RNA כנקודת התחלה, התחלנו עם לנתח האזורים של עניין. בגלל האינטרס שלנו היה גילוי חלבוני phosphorylated שהיו מעיד על טפיחת איתותפעילות hway במהלך תקופה מסוימת של התפתחות שסתום אב העורקים (E13.5-14.5), ביצענו את כל הניתוחים במאגר טריס נטול פוספט ואספתי את השסתומים במאגר תמוגה מבוססת טריס עם מעכבי phosphatase ופרוטאז. במקרה הספציפי שלנו, רק אזורי שסתום אב העורקים נאספו, אבל יכול בקלות להיות מושגת אזור שסתום ריאתי באותו הזמן. אזורי שסתום היו אז הומוגני ושילוב עם מאגר מדגם המשמש כיום ללמוד ביטוי חלבון בשסתומי לב מבוגר 7. על ידי שימוש בכמויות קטנות מדגם (למשל, 2 μl) ואיגום אזורי שסתום מעוברים עם גנוטיפ זהה, שהיינו מסוגל לזהות חלבונים עוברים פוספורילציה וnonphosphorylated ביום עוברי 13.5 8. מאחר שניתן להקפיא את אזורי שסתום ומאוחסנים במאגר תמוגה, ניתן genotyped עוברים במידת צורך לפני איגום. טכניקה זו מרחיבה את הסט של כלים הזמינים להערכת סיגנליזטורי תאמסלולים גרם במהלך פיתוח ומספק מחמאה כמותי לאימונוהיסטוכימיה, במיוחד עבור שסתומי לב מתפתחים. טכניקה זו צריכה להיות בעל ערך לא רק לקרדיולוגים התפתחותיים, אלא גם לכל הביולוגים התפתחותיים שעובדים עם עוברים בראשית התפתחות מעוניינים באזורים המכילים רקמה מוגבלת.

<table border="0" cellpadding="0" cellspacing="0" width="972">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:512px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Timed-pregnant mouse</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td>To be dissected at the embryonic stage of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereoscopic microscope</td>
			<td style="width:125px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.1 M Tris, pH 7.6</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscissors</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:119px;">15003-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fine forceps, #5</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:119px;">11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting needle holders</td>
			<td style="width:125px;">Ted Pella Inc.</td>
			<td style="width:119px;">13560</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting needles</td>
			<td style="width:125px;">Ted Pella Inc.</td>
			<td style="width:119px;">13561-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micropipette, 20 &#956;l, with tips</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysis buffer</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td>50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PhosSTOP</td>
			<td style="width:125px;">Roche</td>
			<td style="width:119px;">4906845001</td>
			<td>Add 1 tablet to 10 ml lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TissueLyser LT</td>
			<td style="width:125px;">Qiagen</td>
			<td style="width:119px;">85600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel beads</td>
			<td style="width:125px;">Qiagen</td>
			<td align="right">69989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

protein isolation from the developing embryonic mouse heart valve region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

שימוש בטכניקת הכנה זו, שהיינו מסוגל לזהות סמאד 1,5,8 פוספורילציה (pSmad) באזורי שסתום אב העורקים אחת מE13.5 עוברים. כפי שניתן לראות באיור 1 א, החלבון מבודד מאף אזור שסתום חד מספיק כדי לזהות להקת pSmad קלושה. עוצמת אות מגדילה באופן יחסי למספר אזורי שסתום שייאגרו. חש...

Watch this Scientific Journal Video about Protein Isolation from the Developing Embryonic Mouse Heart Valve Region at JoVE.com

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

בידוד חלבון מהעכבר עוברי שסתום לב אזור פיתוח

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

protein-isolation-from-developing-embryonic-mouse-heart-valve

Research

JoVE Journal

Biology

9.3K Views.  University of North Carolina at Chapel Hill. The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Video: Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-דיסקציה של מוח החולדה עבור הפקת RNA או חלבון מן אזור מסוים במוח

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

בידוד גזירת תאים עובריים בעכבר ז&#39;רמינל

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

בידוד והתרבות של תאים מאזור nephrogenic של הכליה עכבר עובריים

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Immunostaining הלב גזור עוברי דג הזברה

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

דור של עכברים שמקורם בתאי גזע pluripotent מושרים

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

בידוד תאים כמו Melanocyte-ראשיים מלב העכבר

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

רומן<em&gt; Ex vivo</em&gt; שיטת תרבות ללב העכבר העוברית

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

בידוד של תאי האנדותל חדרית עובריים

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

תיוג תא והזרקה בפיתוח לבבות עכבר עוברי

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

בידוד של תאי Murine Valve האנדותל

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...