Yazarlar (Bölüm Göz ve Görsel Bilimler) Tıp St. Louis Okulu Washington Üniversitesi&#39;nde Fare Genetik Core Moleküler Genetik çekirdek, Morfoloji ve Görüntüleme çekirdeğinin çalışanlarına teşekkür ederiz. Yazarlar Cellular için McDonnell Merkezi ve Moleküler Nörobiyoloji, Sinir Hastalıkları Umut Merkezi&#39;nden küçük hibe programı tarafından desteklenen, Ulusal Göz Enstitüsü (DH # R01EY022632), Ulusal Göz Enstitüsü Merkezi Çekirdek Grant (# P30EY002687) ve Research sınırsız hibe Göz ve Görsel Bilimler Bölümü&#39;ne Körlük Önlemek için.

Etik Beyanı: Hayvan denekleri Prosedürler St. Louis Washington Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. 1. Fare Islahı ve Genotiplendirme <ol><li> (PCP2Cre CAG-EGFP / KASH2) 19,20 Tg üretmek için Tg (CAG-LacZ / EGFP-KASH2) Tg fareler (PCP2-Cre) fareler Breed. Not: Bu protokol kalanı serebellumdaki Purkinje hücreleri EGFP-KASH2 ekspresyonunu sınırlamak için Tg (PCP2-Cre) fare hattı kullanımı üzerinde odaklanmasına rağmen, benzer bir prosedür başka Tg (Cre) fare ile takip edilebilir ( Şekil 2A). </li><li> Daha önce açıklandığı gibi 19 hem EGFP ve Cre transgenini barındıran yavrular tanımlamak için genotipleme gerçekleştirin. </li></ol> Diseksiyon ve Doku Koleksiyonu için Materyallerin 2. Hazırlık <ol><li> -20 ° C&#39;de% 30 PBS sükroz (1x PBS 30 g sükroz / 100 mi) ve deposunun 100 ml hazırlayın. Doku h günlüktranscardial perfüzyonları bir 35 ml şırınganın içine arvesting, çözündürme ve sukroz çözeltisi yük ~ 30-35 ml olmuştur. </li><li> PBS içinde% 4 PFA 40 ml (10 mi,% 16 paraformaldehit ve 30 ml PBS) hazırlayın. Transcardial perfüzyonları için 35 ml şırınganın içine Yük ~ 30-35 ml% 4 PFA. </li><li> Doku diseksiyon 10 ml PBS ile bir 10 cm&#39;lik bir doku kültürü plağı doldurun. </li><li> 16 mg / ml ketamin ve 3 mg / ml ksilazin içeren bir ketamin / ksilazin kokteyli hazırlanır. </li><li> Diseksiyon için% 70 etanol içinde bir çözelti hazırlayın. </li></ol> 3. Diseksiyon ve Doku Koleksiyonu <ol><li> Bir tane 1 ml&#39;lik şırınga kullanılarak, vücut ağırlığının her 25 gramı için, ketamin / ksilazin kokteyli 500 ul enjekte edilir ve 15 dakika ~ bekleyin. Alternatif olarak, ilgi biyolojik sistemine uygun olan başka bir kurumsal onaylı sedasyon yöntemi kullanın. </li><li> Sedasyon üzerine hafifçe dorsal tarafı ile strafor tepsi üzerinde tüm pençeleri pin, ağrı yanıtı eksikliği onaylamak için arka pençe tutamfare aşağı bakacak. </li><li> % 70 etanol ile karın Sprey ve kalp erişmek için göğüs kafesinin üst alt karın bir kesi yapmak. </li><li> Kalbin sağ atrium küçük bir kesi yapmak ve kalbin sol ventrikül içine% 30 sukroz çözeltisi içeren şırınga yerleştirin. </li><li> ~ 2-5 mi başına dakika bir hızda çözelti serpmek. Ne zaman boş şırınga kaldırmak ve% 4 PFA çözümü ile tekrarlayın. </li><li> Strafor tepsiden fare sabitlemesini ve beyin erişmek için başını kesmek. </li><li> Forseps ve makas kullanılarak hayvanın başının etrafında deri kaldırmak. Bir iğne ile kafatasının arka kısmında bir delik Poke ve dikkatli makas ve kesme forseps ile kafatası çıkarın. </li><li> Beyin açıkça erişilebilir bir kez, beyincik kaldırmak ve PBS içinde yerleştirmek için forseps kullanabilir. Bir neşter ile sajital düzlemde beyincik iki eşit parçaya böler ve 4 ° C&#39;de bir gece% 30 sukroz çözeltisi aktarın. Not: hazırlık içinparafin kesitler vardır öncesinde doku gömme ve kesit için% 4 PFA 4 ° C&#39;de gece boyunca beyincik sabitleyin. </li></ol> 4. Doku İşleme ve Kesit <ol><li> Kuru buz ve 2-metil bütan içeren bir bulamaç hazırlanır. Banyosunun sıcaklığı 5 dakika boyunca dengelenmeye bırakın. </li><li> OCT bileşiği ile cryomold doldurun ve kalıba iki eşit parçaya bölünmüş beyincik yarısı yerleştirin. </li><li> Bir diseksiyon mikroskobu ve forseps pozisyon bir çift (beyincik orta hat yakınında) iki eşit parçaya bölünmüş bir yüzey yukarı bakacak şekilde beyincik kullanma. </li><li> Ekim bileşiği, bir doku numunesinin yaklaşık beyaz bir katı matris oluşana kadar 5 dakika boyunca kuru buz bulamaca cryomold aktarın. Not: Gerekirse dondurulmuş numune hemen -20 ° C &#39;de muhafaza edilebilir. </li><li> Etiket cere 15 mikron bölümleri toplamak amacıyla üreticinin talimatlarına göre, doku bölümleri toplamak için poli-lisin slaytlar tedavi edilen ön ve Kriyostat hazırlanmasıbellum. Bölümleri toplamak için dikkatle slayt poli-lizin kaplı tarafı Ekim bölümüne başvurun. Bölümünün yönünü korumak için, bu aşama esnasında slayt dönmez. </li><li> Boyama ve görüntüleme ile devam kadar -20 ° C&#39;de saklayın doku bölümleri. </li></ol> 5. Boyama ve Beyincik Bölümlerin Görüntüleme <ol><li> Doku boyamadan önce aşağıdaki çözümleri hazırlayın: PBS ~ 50 mi ~ PBS içinde% 4 PFA 5-10 mi ~ PBS içinde% 0.5 Triton X-100,% 10 eşek serumu 10-20 mi </li><li> Çözülme serebellar kesitler. Önemli olarak, EGFP solmasının engellenmesi için, sürekli ve yoğun herhangi bir ışık kaynağı maruz kaldıkları en aza indirir. </li><li> Hidrofobik bir bariyer kalemini kullanarak, doku kesitlerinde etrafına bir dikdörtgen çizin. </li><li> 5 dakika boyunca% 4 PFA ile bölümleri Post-fix. Hidrofobik bariyer (genellikle ~ 500 ul) tarafından tarif tüm alanı kapsayacak şekilde yeterli sıvı kullanın. , Bir vakum ya da pipet yeniden kullanmasabitleştirici doku kesitlerinde rahatsız etmemek için özellikle dikkatli olmak taşıyın. </li><li> Bir pipet ile hafifçe doku bölümleri bozmadan slayt PBS uygulanır. 5 dakika sonra dikkatlice sıvıyı çıkarmak için vakum veya pipet kullanın. PBS üç kez toplam durulama tekrarlayın. </li><li> Bir pipet kullanarak dikkatli bir şekilde, PBS içinde% 0.5 Triton X-100,% 10 eşek serumu (çözeltisi 900 ul başına 3 ul kalbindin ve / veya Nesprin2 antikoru) uygun bir primer antikor seyreltme uygulanır. Karanlıkta, oda sıcaklığında bir saat süreyle inkübe edilir. Not: EGFP-KASH2 dondurulmuş kesitler üzerinde doğrudan floresan mikroskobu ile görünür olacaktır, ve bir anti-EGFP antikor kullanılması gerekli değildir. Parafin kesitleri hazırlanmış, ancak EGFP-KASH2 görüntüleme, bir anti-EGFP antikor ile immunodeteksiyon gerektirir. </li><li> Antikor çözüm çıkarın ve PBS ile üç kez yıkayın. </li><li> 1 Hazırlama:% 0.5 Triton X-100 içinde Alexa Fluor sekonder antikor seyreltileri 1000,Karanlıkta% 10 eşek PBS serum ve mağaza. </li><li> Yavaşça Nihai PBS durulama çıkarın ve karanlıkta bir saat süreyle ikincil antikor seyreltme uygulanır. </li><li> Ikincil antikor çözüm çıkarın yıkayın ve bölüm üç kez yıkayın. Gerekirse, ilk PBS uygulama esnasında, örneğin DAPI (~ 300nM) olarak nükleer leke, uygulanır. </li><li> Tamamen doku ve doku bölümünde medya montaj yeri ~ 5-7 ul yerinden PBS çözüm kaldırmak. Doku bölümünün üstünde bir cam lamel yerleştirin ve karanlıkta, oda sıcaklığında ~ 15-30 dakika boyunca bekletin. </li><li> EGFP ve adımları 5,8 / 5,9 kullanılan ikincil antikorlar için uygun olan herhangi bir başka dalga boylarını görselleştirmek için uygun filtreler ile donatılmış bir fluoresans mikroskopu kullanın. Serebellar dilimleri, EGFP-KASH2 nükleer jantlar görüntülemek için bir 20X objektif kullanın. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

En kritik aşama başarılı Tg (CAG-LacZ / EGFP-KASH2) modeli, uygun bir Cre fare hattı (ler) i belirlemek için kullanıldığı in vivo LINC komplekslerinin rolünü incelemek için. Cre içindeki yollar yer alan diğer hücre tiplerinde aktif Gerçekten de, eğer, bu sonuçların yorumunu komplike olabilir. Beyincik (Şekil 2B), moleküler ve granül hücre katmanlarında burada gösterildiği gibi Dolayısıyla, bu, at hücreleri incelemek için önemlidir. Aynı şekilde, herhangi bir fi...

Nükleer zarf (NE) Sitoplazmadan nükleolazma ayırır. Nükleer gözeneklerin bağlamak bir iç ve bir dış çekirdek zarı (INM sırasıyla Onm,) oluşmaktadır. Her iki membranlarla tarif lümen perinükleer alanı (PNS) olarak adlandırılır. Onm granüllü endoplazmik retikulum (ER) bir uzantısı olan ve INM nükleer lamina yapışır, A ve B tipi ile temsil edilen çekirdek tip V ara ipliklerin bir ağ örgüsü 1,2 lamins. Nucleoskeleton ve hücre iskeleti (LINC) komplekslerinin bağlayıcılar fiziksel iskelet ipliklerin ve moleküler motorlar (Şekil 1A) çekirdeğin iç bağlamak için bütün çekirdek zarfı kapsayan makromoleküler monte edilmiş halleridir. Güneş (Sad1 / Unc84) proteinler ve Nesprins (Nükleer Zarf SPectRINS): Bunlar KD ayrılmaz transmembran proteinlerinin iki aileyi karakterize evrimsel korunmuş motiflerinin arasındaki etkileşimleri oluşur. Memelilerde, Sun1 ve Sun2 arN-terminal bölgesi nucleoplasmic A ve B tipi lamins 3-5 ile doğrudan etkileşip etkileşmediği INM e transmembran proteinleridir. PNS içinde INM diğer tarafında, Sun proteinler 150 C-terminal amino asitler, SUN alanı adı verilen ~ evrimsel korunmamış streç barındırır. SUN etki evrimsel korunmuş Kash ile doğrudan etkileşim (Klarsicht / Anc-1, Syne Homology) etki, Nesprins moleküler imza. Kash etki, bir zar geçiş alanı 6 ve ardından PNS içine çıkıntı ~ 30 C-terminal amino asidi bir streç oluşur. En az dört farklı Nesprin genler (Nesprin1-4) NE 7 lokalize proteinler Kash içeren kodlar. Şaşırtıcı bir 1000 kDa (Nesprin1 dev) için boyutları değişir ~ 50kDa (Nesprin4) den Nesprins, sitoplazmik bölgeleri, çoklu spektrin gibi aktin, plectin ve moleküler motorlar 8- olarak iskelet bileşenleri ile etkileşimi sağlayarak tekrar yanı sıra özel motifler içeriyor 13. &lt;omurgalı ve omurgasız p class = &quot;jove_content&quot;&gt; Araştırmalar Lamin / Güneş / Nesprin / moleküler motorlar bir evrimsel olarak korunmuş &quot;eksen&quot; nükleer göç ve ankraj kontrol teşkil ettiğini göstermiştir. Linc karmaşık bileşenlerin çeşitli knock-out (KO) fare modelleri tanımlanmış ve memeli gelişim 9,14,15 sırasında KD Sun ve Nesprin proteinlerinin rollerini anlamak için bir çerçeve sağlayan aracı vardı oylandı. Ancak, bu modeller en önemlisi mevcut birçok önemli sakıncaları: of fenotipik katkılarını ayırt olmayan özerk etkileri, 2) zorluk hücre nedeniyle fenotipleri yorumlarken 1) zorluk Kash-az Nesprin vs Kash içeren, 3) 16 izoformları Çok sayıda hücre tiplerinde NE Sun ve Nesprin proteinlerin işlevsel fazlalık hem Kash-etki alanı için eksik farelerde 17 tüm SUN-Kash etkileşimleri ve farelerin 4) perinatal öldürücülüğü inaktive karmaşık ıslah planları gerektirirNesprins1 ve 2 yetişkin analizi 18 fenotip engellemektedir. Bu protokol, böylece yukarıda özetlenen sakıncaları birçok atlayarak bir hücre özerk ve gelişimsel olarak düzenlenen bir şekilde in vivo tüm SUN-Kash etkileşimleri bozmak için tasarlanmış yeni bir fare modeli açıklanmaktadır. Bu Cre / lox tabanlı fare modeli, iki önemli kavramları dayanır: 1) bilinen herhangi bir Nesprin proteininin Kash alanı hücre kültür sistemlerinde KD EGFP hedef ve 2) SUN etki fazla sentezlenmesi, böylece, Kash etki alanları ile etkileşime promiscuously için yeterlidir Herhangi Kash etki tüm endojen Paz etki doyurmak ve dominant-negatif bir şekilde 17 (Şekil 1B) Linc kompleksleri inaktive edecek. Bu protokol, doku hasat ve serebellar Purkinje hücrelerinde tüm SUN-Kash etkileşimlerinin bozulması doğrulamak için kullanılan işlem adımları açıklar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion Slides</td>
			<td>StatLab</td>
			<td>M1000W</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Sigma Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmuEdge Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Calbindin Antibody</td>
			<td>Sigma Aldrich</td>
			<td>C9848</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-EGFP Antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nesprin2 Antibody</td>
			<td></td>
			<td></td>
			<td>Previously described in Ref. 21</td>
		</tr>
		<tr>
			<td>Fluorescent Mounting Media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>Sigma Aldrich</td>
			<td>O3551</td>
			<td></td>
		</tr>
	</tbody>
</table>

validation of a mouse model to disrupt linc complexes in a cell-specific manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Bu protokol, Tg (PCP2-Cre) fareler kullanılarak beyincik Purkinje hücreleri için EGFP ekspresyonu KASH2 kısıtlamak için Tg (CAG-LacZ / EGFP-KASH2) fare modelinde yararını göstermektedir. Tg olarak (PCP2Cre CAG-EGFP / KASH2) yavruları, LacZ / V5 açık okuma çerçevesi bu şekilde özellikle Purkinje hücreleri (Şekil 2A) için hedeflenen EGFP-KASH2 ekspresyonuna yol açan Cre rekombinaz tarafından P6 eksize edilir. Beklendiği gibi çekirdeğe (Şekil 2B) çevres...

Watch this Scientific Journal Video about Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner at JoVE.com

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Fare Modeli Doğrulanması Hücre-spesifik şekilde LINC kompleksler bozacak

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

validation-mouse-model-to-disrupt-linc-complexes-cell-specific

Research

JoVE Journal

Biology

7.3K Görüntüleme.  Washington University in St. Louis School of Medicine. Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Video: Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Potansiyel Parkinson Hastalığı Genler Doğrulama için C. elegans Dopamin Nöron Dejenerasyonu Testi Uygulama

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Biyoloji

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-iç hücre embriyonik kök hücrelerin kitle enjeksiyon daha yüksek Chimerism oranları için yol açar.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Fluorescently etiketli CXC Kemokin Ligand 12 CXC Kemokin reseptör 4 için bağlama bozabilir bileşikler tanımlamak için Akış Sitometresi tabanlı tahlil

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Bağırsak kısmi tıkanıklık bir fare modeli

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP etiketli CENP-A (EYFP-CENP-A) Ubiquitylation tanımlamak için Kütle Spektrometresi Analizi

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Bel Omurgası Kararsızlığının Fare Modeli

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Primer Kültürde Diferansiye Fare Adipositleri: İnsülin Direncinin Bir Modeli

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...