עבודה זו נתמכה על ידי מענקים לJLRR מ: אוטיזם ספיקס, נינה אירלנד, ווסטון נמלי קרן, NIMH R01 MH081880, וNIMH R37 MH049428. PRW נתמכה על ידי מענק מהמועצה הלאומית למדע של טייוואן. SFS נתמכה על ידי F32 (MH103003).

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The authors declare that they have no competing financial interests.

השימוש של מבשרי interneuron קליפת המוח GABAergic מהוד ganglionic העוברי (GES) לטיפולים בתאים מבוססים מראה הבטחה למצבים רבים 12 -1 4. יש צורך בטכניקות מולקולריות מדויקות כדי לעקוב אחר, ולהביע את הגנים של עניין בתת-קבוצות interneuron ספציפיות. כאן אנו מספקים פרוטוקול מפורט לתיוג תאים עוב?...

interneurons קליפת המוח GABAergic מהווה ~ 20-30% מתאי עצב שבקליפת המוח של היונקים, ואילו השאר מתאים לגירוי, הנוירונים עיקריים glutamatergic. Interneurons מגוונת מאוד בנכסים, האקסון אלקטרו ומורפולוגיה דנדריט והסינפטי מיקוד 1, וחוסר איזון בטון מעכב / מעורר הם שיערו לבסיס כמה פנוטיפים של הפרעות נוירו-פסיכיאטריות / נוירולוגית כוללים אוטיזם, סכיזופרניה ואפילפסיה 2. המטרה הכוללת של הפרוטוקול המתואר במסמך זה היא לספק אמצעים לשנות ביעילות גנטית אבות interneuron קליפת המוח GABAergic לפני ההשתלה לin vivo ניתוחים. interneurons קליפת המוח GABAergic נולדות בהוד המדיאלי וזנב ganglionic (MGE וCGE, בהתאמה) 3,4, כמו גם את אזור הקדם-האופטי התיכון 5. אבות interneuron קליפת המוח עוברים הגירה משיקה למרחקים ארוכים ואחריעל ידי הגירת רדיאלי להגיע ליעדים הסופיים שלהם. עם הגעה ליעדים שלהם, interneurons קליפת המוח אלה חייבת בצורה נכונה להשתלב ברשת העצבית הקיימת, וכל תת-קבוצה interneuron ייחודית תתרום למעגלים בקליפת המוח בדרכים מסוימות. ניתן להבחין בארבע קבוצות עיקריות על ידי סמנים מולקולריים: סומטוסטטין נגזר MGE (SST) + וparvalbumin (PV) + תת-קבוצות, ופפטיד נגזר CGE vasoactive מעיים (VIP) + וReelin +; SST - תת-קבוצות 6. תת-קבוצות שונות בקליפת המוח interneuron נולדות על זמנים שונים במהלך התפתחות עוברית ובMGE 7 CGE, 8. אלה וסמני interneuron אחרים בקליפת המוח GABAergic כבר השתמש כדי ליצור קווי Cre-נהג ספציפיים עבור רבים מתת-קבוצות אלה 9-11. ההשתלה של תאי אב MGE התפתחה כטיפול מבוסס תאי פוטנציאל לטיפול בהפרעות שעלולות להיגרם על ידי חוסר איזוןבעירור / עיכוב 12-24. יתרונות טיפוליים אלה עשויים להיות נובעים בחלקו את היכולת הייחודית של אבות MGE (לפיזור, להבדיל ולהשתלב במוח מארח), או שעלול להיות בגלל פרי גופני רב PV + תאים מעכבים נגזרים מMGE. תאי MGE יכולים להיות גם transduced מהירות וביעילות עם lentiviruses לפני ההשתלה 15, המאפשרים לתאים שעברו שינוי גנטי במבחנה כדי להיחקר in vivo. הרציונל לפיתוח גישה זו היה להתגבר על מחסומים בלימוד פיתוח GABAergic קליפת המוח interneuron והתבגרות. בפרט, השתלת MGE אפשרה לחוקרים ללמוד את התפתחותם של תאים שעברו מוטציה in vivo, כאשר עכבר המוטציה אחרת היה מת בנקודת זמן מוקדמת. יתר על כן, על ידי החדרת גנים של עניין לפני ההשתלה, את ההשפעות של גנים ספציפיים על פנוטיפ מוטנטי יכולות להיות מוערכים בEFFאופן icient. כאן, אנו מספקים פרוטוקול מפורט לtransduce תאי MGE עם lentiviruses לפני ההשתלה. בנוסף, אנו מראים כיצד טכניקה זו ניתן להתאים להביע גן של עניין בתת-קבוצות interneuron ספציפיות מקבוצה הטרוגנית של מבשרי interneuron קליפת המוח, באמצעות שילוב של lentiviruses הביטוי Cre תלוי וקווי עכבר Cre-נהג זמינים. יתר על כן, פרוטוקול זה מציג טכניקות ופלטפורמה לחוקרים לשנות גנטי מבשרי interneuron קליפת המוח GABAergic לin vivo מחקרים בדרך ייחודית. אחד יתרונות של שיטה זו על פני גישות שוטפות אחרות הוא שהתאים המושתלים MGE יתפזרו מאתר ההזרקה. כמו כן, בניגוד לזריקות נגיפיות מוקד, לאחר תאי MGE לפזר המורפולוגיה שלהם קלה יותר להעריך. גישה זו יכולה לשמש כדי לחקור את ההשפעה של החדרת גנים של עניין לסוג בר או תאים שעברו מוטציה, מציג סוג תא מסויםכתב להעריך מורפולוגיה, או שעלול להיות כדי לחקור את ההשפעה של אללים מחלה in vivo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and equipment for MGE cell transplantation</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>REF 309602</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>301/2 gauge needle</td>
			<td>305106</td>
			<td colspan="2">BD, Becton Dickinson and company</td>
		</tr>
		<tr>
			<td>Precision bore to deliver 5 &#181;l (comes with plunger)</td>
			<td>5-000-1005</td>
			<td colspan="2">Drummond scientific company</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PM-999</td>
			<td colspan="2">Polysciences, Inc.</td>
		</tr>
		<tr>
			<td>Stereo microscope with boomstand **</td>
			<td>MZ6</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital just for mice stereotaxic instrument **</td>
			<td>51725D</td>
			<td colspan="2">Stoelting company</td>
		</tr>
		<tr>
			<td>Single-axis oil hydraulic fine micromanipulator **</td>
			<td>MO-10</td>
			<td>Narishige</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond coated rotary beveler</td>
			<td>n.a.</td>
			<td colspan="2">made in house</td>
		</tr>
		<tr>
			<td>Needle pipette puller</td>
			<td>730</td>
			<td colspan="2">Kopf instruments</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>n.a.</td>
			<td colspan="2">Any drug store or pharmacy</td>
		</tr>
		<tr>
			<td colspan="2">Any pipette and tips that can reliaby measure 1 &#181;l of volume</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">** These are the particular models we use, but many other setups should work</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Optional Reagents (for making Lentiviruses)</td>
		</tr>
		<tr>
			<td>25 mm, 0.45 &#181;m filter</td>
			<td>09-719B</td>
			<td colspan="2">Fisher Scientific</td>
		</tr>
		<tr>
			<td>Ultra-clear centrifuge tubes (25 x 89 mm)</td>
			<td>344058</td>
			<td colspan="2">Beckman Coulter&#174;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (1.5 ml size)</td>
			<td>11668019</td>
			<td colspan="2">Invitrogen</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other commercially available supplies</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE plasmid encoding gag/pol</td>
			<td>12251</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-Rev plasmid encoding Rev</td>
			<td>12253</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G plasmid encoding VSVG</td>
			<td>12259</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-Flex-GFP</td>
			<td>28304</td>
			<td>Addgene</td>
			<td></td>
		</tr>
	</tbody>
</table>

viral-mediated labeling and transplantation of medial ganglionic eminence (mge) cells for in vivo studies

interneurons GABAergic קליפת המוח, הנגזרת מהמדיאלי העוברית והוד הזנב ganglionic (MGE וCGE), היא מבחינה תפקודית ומורפולוגית מגוונת. אחיזה נעשתה בהבנת התפקידים של תת-קבוצות שונות בקליפת המוח interneuron, עם זאת, עדיין יש מנגנונים רבים יש לעבד שעשויה לתרום להתפתחות והתבגרות סוגים שונים של תאי GABAergic של. יתר על כן, איתות GABAergic שינתה עשויה לתרום לפנוטיפים של אוטיזם, סכיזופרניה ואפילפסיה. קווי Cre-נהג ספציפיים החלו לעטוף את הפונקציות של תת-קבוצות interneuron ייחודיות. למרות ההתקדמות במודלים של עכברים, לעתים קרובות קשה ללמוד אבות interneuron קליפת המוח GABAergic ביעילות עם גישות מולקולריות in vivo. טכניקה חשובה אחד משמשת ללמוד תכנות האוטונומי התא של תאים אלה היא השתלה של תאי MGE לקליפת מוח מארח. תאים מושתלים אלו נעים בהרחבה, להבדיל,nd תפקודי לשלב. בנוסף, תאי MGE ניתן transduced ביעילות עם lentivirus מייד לפני ההשתלה, ומאפשרים למספר רב של גישות מולקולריות. כאן אנו פירוט פרוטוקול לtransduce תאי MGE ביעילות לפני ההשתלה לניתוח in vivo, באמצעות קווי Cre-נהג זמינים ווקטורי ביטוי Cre-תלויים. גישה זו יש יתרון משום שהוא משלב מניפולציה גנטית מדויקת עם היכולת של תאים אלה לפיזור לאחר השתלה, מאפשר רזולוציה מסוימת תאים מסוג גדולה יותר in vivo.

מאז יש לי תאי MGE היכולת הייחודית להעביר ולשלב כאשר הושתלו הניאוקורטקס מארח 16, שהם מספקים מערכת מודל מצוינת למניפולציה גנטית לפני מחקרי in vivo. במסמך זה, אנו מראים כיצד ניתן לבודד את רקמת MGE מעוברי E13.5 (איורים 1 ו -2), אשר לאחר מכן ניתן transduced עם lentiviruses או <em...

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Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

אבות interneuron קליפת המוח GABAergic לפזר, לפתח וsynaptically להשתלב קליפת מארח לאחר השתלה. ניתן transduced תאים אלה בקלות לפני ההשתלה ללימודי in vivo של מבשרי GABAergic מהונדסים גנטי. כאן, אנו מציגים טכניקות תיוג נגיפיות למקד תת-קבוצות interneuron ספציפיות באמצעות קווי Cre קיימים וכתבי Cre-תלויים.

תאי תיוג ותיווך ויראלי השתלה של המדיאלי ganglionic רוממות (MGE) ל<em&gt; In vivo</em&gt; לימודים

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically ...

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Developmental Biology

Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

11.4K Views.  University of California San Francisco.  אבות interneuron קליפת המוח GABAergic לפזר, לפתח וsynaptically להשתלב קליפת מארח לאחר השתלה. ניתן transduced תאים אלה בקלות לפני ההשתלה ללימודי in vivo של מבשרי GABAergic מהונדסים גנטי. כאן, אנו מציגים טכניקות תיוג נגיפיות למקד תת-קבוצות interneuron ספציפיות באמצעות קו...

Video: Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence MGE Cells for In Vivo Studies

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

שיטה יעילה להשיג תאי Dedifferentiated שומן

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

מחקרי מבנה-תפקוד תאי גזע עוברי עכבר שימוש recombinase בתיווך Exchange קסטה

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

כל תא תיקון מהדק הקלטות של תאים אפיתל העיקרי מבודדים מן האברדימיס

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

רומן חדש פאט שומן טכניקת השתלת לדמיין את כלי השיט הדור של כלי הדם בתאי גזע אנדותל

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

אופקי הר שלם: עיבוד רומן פרוטוקול הדמיה על רקמה עבה, תלת מימדי חתכים של העור

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

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 ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Photoconversion תא בודד בהמחיה שלם דג זברה

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Transcriptomic תא בודד ניתוחים של תאים בלבלב האנדוקרינית העכבר

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

תוך ורידי, אינטרה-שפיר בתוך הרחם השתלת במודל מאתר

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

בדור חוץ גופית של Somite נגזרים מן האדם המושרה בתאי גזע Pluripotent

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

תאי תיוג ותיווך ויראלי השתלה של המדיאלי ganglionic רוממות (MGE) ל

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