Yazarlar hiçbir çıkar çatışması olarak atadım.

Genel protokol sepsis indüksiyon ilk gününde ve yalıtım BAL sıvı hayvan ve fare BALF exosomes izolasyonu için ikinci gün de dahil olmak üzere 2 gün gerektirir. Tüm yordamları gözden geçirilmiş ve kurumsal hayvan bakım ve kullanım Komitesi Atlanta VA Tıp Merkezi tarafından onaylanmış.
1. fare Akut Septik akciğer yaralanma modeli
<ol><li>Kullanın 6-8 hafta - yaşlı erkek yaban-C57BL/6J farelerde tip (ağırlığı 20-22 g) hayvan için model ve aseptik teknik ve araçları kullanarak tüm yordamlarını gerçekleştirin. Otoklav forseps ve makas için 121 ° C'de 20 dk da dahil olmak üzere tüm cerrahi aletler</li>
	<li>Rastgele fare içine deney ve kontrol grubu dağıtın. Fareler cerrahi bir tahta üzerinde tut. Escherichia coli lipopolysaccharide (LPS, 15 mg/kg) ile fareler mayi yoldan tedavi. Bireysel vücut ağırlığına göre uygun LPS dozlarda fare, başına 0.1 ml PBS sulandırmak LPS çözüm 27 G iğne ile 1 mL şırınga kullanarak enjekte ve PBS bir eşdeğer birim denetim hayvanlara enjekte.</li>
	<li>Fareler uygun oda sıcaklığında temiz kafes oluşur hayvan BSL2 tesis unutmayın.</li>
	<li>24 s sonra CO2 inhalasyon ile fareler ötenazi.</li>
	<li>Cerrahi alan fare boyun ortasına seçin. Küçük bir insizyon attım (yaklaşık 5 mm) yapmak ve yavaşça kasları erişim trakea maruz kadar künt forseps kullanarak nefes borusu için ayırın.</li>
	<li>Fare BAL sıvısı 10 mL steril şırınga ile 27 G iğne ile geri alıyorum. İlk olarak, yavaşça, iğne nefes borusu takın ve 1 mL steril PBS yavaş yavaş nefes borusu enjekte, daha sonra BALF 3 mL tek kullanımlık şırınga 27 G iğne ile çizmek. Yordamı tamamlandığında, şırınga ve iğne biohazard "Sharps" kap içinde atın.</li>
	<li>Chauret ve ark. tarafından daha önce açıklandığı gibi cerrahi aletler ile klor dioksit, tedavi 27ve o zaman ısıyla 121 ° C'de 20 dk için tarafından tüm cerrahi aletler sterilize</li>
</ol>2. Exosomes yalıtım-seri Ultra-Santrifüjü ve karakterizasyonu
<ol><li>Fare BAL sıvısı 15 mL konik tüpler toplamak. Alveolar hücre hasat için örnekleri, 1000 g, 4 ° C'de 10 dakika santrifüj kapasitesi</li>
	<li>Süpernatant yeni 15 mL konik tüpler, daha sonra örnekleri için 4.000 x g 4 ° C'de hücre artıkları kaldırmak için de 30 dk santrifüj kapasitesi.</li>
	<li>Süpernatant ultracentrifuge tüpler ve 10.000 x g 4 ° C'de daha büyük hücresel parçacıkları kaldırmak için de 60 dk santrifüj örnekleri toplamak.</li>
	<li>Süpernatant yeni konik tüpler, daha sonra örnekleri 0.2 µm şırınga filtre daha büyük partiküller kalan kaldırmak için aracılığıyla filtre.</li>
	<li>Ultracentrifuge tüpler örnekleri toplamak ve tüpler denge. 140.000 x g 4 ° C'de exosomes cips için de 2 h için örnekler santrifüj kapasitesi. Not: 20-200 nm exosomes boyutu değişir. 0.2 µm filtre 200'den fazla olan büyük parçacıklar kaldırmak için kullanılır nm boyutunda.</li>
	<li>Supernatants yavaşça atmak. Pelet 100 µL PBS veya lizis çözüm ve transfer exosomes örnekleri için 1,5 mL tüpler ile dağıtılması ve-80 ° C'de kullanmak kadar saklamak. Not: Transmisyon Elektron mikroskobu izole exosomes doğrulamak için önerilir.</li>
	<li>İletişim kuralı28işleme standart TEM örnek izleyin. 200 bar transmisyon elektron mikroskobu ve almak görüntüleri, ölçek kullanarak exosomes örnekleri analiz nm.</li>
</ol>3. miRNA ve sahne miRNA-q-RT-PCR hazırlanması
Not: miRNA-q-RT-PCR miRNAs ifade düzeyini analiz etmek için önerilir.
<ol><li>Standart miRNAs yalıtım Protokolü izleyin ve tek iplikçikli Ters transkripsiyon24saat içinde arıtılmış RNA'ların eşit miktarda kullanmaya dikkat edin.</li>
	<li>Exosomal mikroRNA düzeyi BAL sıvısı içinde algılamak için standart gerçek zamanlı PCR Protokolü izleyin. Belirli gerçek zamanlı PCR astar miR-155, miR-146a ve U6 SnRNA karşı kullanın. PCR reaksiyon kantitatif PCR aparatı ile çalıştırın ve iç kontrol U6 snRNA için ifade değerleri normalize. Not: Her grup onaylatılacak örnekleri24toplam oluşur.</li>
</ol>4. Exosomes etiketleme ve arıtma
<ol><li>Exosomes Pelet 100 µL süspansiyon çözüm ile 1,5 mL polipropilen boru içinde askıya sonra exosomes boya diğer tüp içine 0.4 µL süspansiyon çözeltinin 100 µL ile ekleyin.</li>
	<li>Hızlı bir şekilde exosomes boya çözümde ekleyin ve karıştırarak iyice pipetting tarafından. Örnekleri 5 min için oda sıcaklığında tutmak, ışıktan korumak.</li>
	<li>Bir eksozom spin sütuna bir 1,5 mL elüsyon tüpü yerleştirin. Çözüm karışımı eksozom spin sütun Center'a aktarın.</li>
	<li>Sütun vasıl 800 x g oda sıcaklığında 2 min için spin.</li>
	<li>Sütun atın ve -20 ° C'de eluted örnek kullanmak kadar saklamak.</li>
</ol>5. doğrulama Exosomes alıcı hücreleri tarafından alınan up
<ol><li>1 x 104 MLE 12 /cell bir 8-şey odası slayt ve kültür hücreleri 37 ° c, % 5 CO2tohum.</li>
	<li>10 µL floresan etiketli exosomes hücrelere ekleme. 37 ° C'de 1 h için kuluçkaya</li>
	<li>Kültür ortamını çıkarın ve hücreleri PBS ile yıkayın, sonra oda sıcaklığında 10 dakika için %1 paraformaldehyde ile hücreleri tamir.</li>
	<li>Slayt Dağı orta DAPI ile bağlayın.</li>
	<li>Floresan mikroskop örnekleri analiz ve fotoğraf, 40 X büyütme çekmek.</li>
</ol>6. Exosomes ve alıcı hücreler arasındaki etkileşim doğrulanması
<ol><li>Tohum 2 x 105 MLE12/iyi uygun kültür orta ve 37 ° c, % 5 CO2kültür hücreleri içeren bir 12-şey kültür plaka.</li>
	<li>20 µg exosomes fare BAL sıvısı MLE12 hücrelere ekleme ve deney grubu başına çoğaltır ayarlayın.</li>
	<li>24 s sonra PBS hücrelerle yıkama ve 600 µL lizis arabellek kuyunun içine ekleyin. Yavaşça plaka sallamak ve pipet ile 1,5 mL tüp lizis örnekleri toplamak.</li>
	<li>Standart toplam RNA izolasyon iletişim kuralı ve cDNA Ters transkripsiyon protokolü takip et, tek iplikçikli cDNA Ters transkripsiyon29tamamlamak.</li>
	<li>MRNA ifade düzey alıcı hücreler29algılamak için standart gerçek zamanlı PCR Protokolü izleyin. TNF-α, Il karşı belirli gerçek zamanlı PCR astar kullanın-6 ve ZO-1. PCR reaksiyonları kantitatif PCR aparatı ile çalıştırın ve iç kontrol GAPDH için ifade değerleri normalize. Not: Her grup onaylatılacak örnekleri oluşur.</li>
</ol>

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Bu eser VA Merit İnceleme 2I01BX001786-06A1 RS için fon tarafından desteklenmiştir.

Fare modelleri hastalıkların belirli genler fizyolojik fonksiyonun değerlendirildiği ve deneme2maliyetini azaltmak için yaygın olarak kullanılır. Açıklanan Akut Septik akciğer hasarı burada inflamatuar yanıt ARDS ile insanlarda görülen taklit eder. Bu model moleküler patogenezi, biyolojik gelişimi araştırmak için ve potansiyel yeni tedaviler5test etmek için geçerlidir.
Ortak kültür sistemleri in vivo çalışmalar...

ALI ve ARDS yaklaşık 1 milyon kişi dünya çapında etkiler sigara kardiyojenik pulmoner ödem tarafından neden olduğu ciddi hipoksemi ile solunum yetmezliği hayati formları vardır her yıl1. ARDS etyolojisinde doğrudan yaralanma için akciğer enfeksiyonları veya aspirasyon ve dolaylı hakaret çeşitli içerir. Son on yılda ARDS moleküler patogenezi artan bir anlayış,2,3için geliştirilen henüz ancak, ARDS için hedeflenen spesifik tedaviler vardır.
Akut akciğer yaralanma birkaç hayvan modeli sağlayan bir köprü deneysel terapiler insan çalışmaları4,5' e çevirmek için geliştirilmiştir. Yaygın olarak kullanılan modelleri yerel yükleme oleik asit, bakteri, LPS ve bleomycin içerir. Diğer bir yaklaşım iskemi-reperfüzyon, cecal tüp ligasyonu ponksiyon, mekanik havalandırma kaynaklı streç yaralanma, hyperoxia veya bakteri ve LPS5sistemik yönetimini içerir. Bu modeller klinik hipotezler test etmek için yararlı bir biyolojik sistem sağlamak ve olası terapiler gelişimi için. İnsan ARDS benzetimini yapmak için hayvan modelleri inflamasyon ve akciğerlerde bariyer işlevindeki kusur ile epitel ve endotel hücrelere akut yaralanma çoğaltmak.
Exosomes arası hücresel doku microenvironment moleküler kompozisyonu havalesi yoluyla iletişimde kolaylaştırmak ve membran veziküller ile 20-200 nm çapında, moleküler içeriği protein, DNA, RNA ve lipidler içerir, vardır. Exosomes hücrelerin endotel hücreleri, epitel hücreleri, düz kas hücreleri ve tümör hücreleri, gibi birden çok türü tarafından salgılanan ve insan vücut sıvısı içinde adlı biri yok. Exosomes çapraz-hadis bağışıklık hücreleri ve stromal hücreler arasında bulaşıcı ve steril inflamatuar hastalıklar sırasında düzenleyen ve çeşitli doğal ve deneysel uyaranlara tarafından fizyolojik sırasında düzenlenmiş olması anormal salınımlarını görünür çalışmalar göstermektedir ve patolojik süreçler6. Böyle iletişim ağı akciğer hastalıkları patogenezinde önemli bir rol oynayabilir ve patofizyolojik ilerleme7,8etkileyebilir. Kodlamayan RNA'ların, miRNAs ve doku ve vücut sıvıları, plazma, sera var mRNA ifade translasyon düzeyinde9,10modüle olarak 18-22 nükleotit.
Farklılaşma ve işlevi hücre birden çok türünün paketlenmiş miRNAs exosomes etkisi ve aşırı düzeyleri hastalıkları, kanser, akciğer hastalıkları, obezite, diyabet ve kardiyovasküler hastalık11de dahil olmak üzere çeşitli ile ilişkilidir, 12,13,14,15,16. Alıcı hücreler ve exosomal miRNAs mekik içine girişini kolaylaştıran hücreler arası iletişim microenvironment17,18hemostaz değiştirme. Akut akciğer-yaralanma bir karmaşık süreçler, birden çok hücre tipleri ile exosomes8geniş hücreler arası iletişim ile ilgili olduğunu. miR-155 ve miR-146a ortak transkripsiyon düzenleyici mekanizma paylaşmak ve inflamatuar yanıt ve immün tolerans19,20katkıda bulunur. Son yıllarda yapılan çalışmalarda her ikisi de exosomal miRNAs bağışıklık hücreleri21arasında mekik ile inflamatuar yanıt modüle gösterir. Ancak, düzenleyici etkileri exosomal miRNAs endotoksin alveoler yanıt temel moleküler mekanizmaları belirsiz kalır, hiç şüphesiz soruşturma potansiyel klinik önemi ve çevirim dolaylı hak daha fazla.
Ortak kültür modelleri inflamasyon ve kanser22,23gibi karmaşık ortamında belirli hücre tipleri arasındaki etkileşimin tanımlamak için istihdam edilmektedir. Bu platformlar özellikle bağışıklık ve yapısal hücreler için hücre tipleri arasında çapraz konuşma sorguya çekmek için alternatif bir stratejisi sağlayabilir.
Intra-trakeal, solumuş, mayi veya sistemik LPS deneysel akciğer hasarı24,25,26ikna etmek için yaygın olarak kullanılan ve yönetim epitel ve endotel geçirgenliği ikna etmek için göstermiştir kusur. Burada mayi LPS septik manken akut akciğer yaralanma farelerde ikna etmek için kullanın. LPS mayi yönetim 24 h içinde geçirgenliği kusurları ile inflamatuar hücre alımı akciğerlerde indüklenen vardır. Ayrıca, exosomes BAL üzerinden miRNA-155 ve miR-146a içerir ve exosomes BAL sıvısı üzerinden alıcı epitel hücreleri, IL-6 ve TNF-α gibi ifadede pro-inflamatuar sitokinlerin neden göster. O exosomal miRNAs ilk Akut Septik akciğer hasarı bu modelde BAL içinde salgılanan verilerdir.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:691px;">lipopolysaccharide</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:224px;">Escherichia coli 055:B5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS pH7.2(1X)</td>
			<td>Life technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mirVana miRNA isolation kit</td>
			<td>Thermo Fisher</td>
			<td align="right">449774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>Thermo Fisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mmu-miR-155 (002571) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">has-miR-146a (000468) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U6snRNA primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TNF-&#945;(Mm00443258_m1 ) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IL-6 (Mm00446190_m1&#160;&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZO-1 (Mm00493699_m1&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minimal essential medium (MEM)</td>
			<td>Thermo Fisher</td>
			<td>11095-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trysin-EDTA solution(0.05%)</td>
			<td>Thermo Fisher</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome Spin Columns (MW3000)</td>
			<td>Thermo Fisher</td>
			<td align="right">4484449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Optima L-100XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-clear centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td align="right">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Applied Biosystems 7500 Fast Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transmission electron microscopes (TEM)</td>
			<td>JEOL Ltd</td>
			<td>120 kV TEM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus BX41 microscope</td>
			<td>Olympus</td>
			<td>BX41</td>
			<td></td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

Akut akciğer hasarı (ALI) ve Akut Respiratuar distress Sendromu (ARDS) yüksek morbidite ve mortalite bulunmaya devam ediyor akciğer hastalıkları heterojen bir grup temsil eder. ALI moleküler patogenezi daha iyi tanımlanmış; Ancak, hastalık karmaşık yapısı nedeniyle moleküler terapiler geliştirilecek için henüz. Burada bir lipopolysaccharide (LPS) indüklenen fare modeli Akut Septik akciğer yaralanma exosomes inflamatuar yanıt rolü betimlemek için kullanın. Bu modeli kullanarak, biz mayi LPS maruz fareler exosomes içinde miRNA ve inflamatuar yanıt düzenleyen sitokinler ile paketlenmiş akciğerler solunum-alveoler lavaj (BAL) sıvı salgılar göstermek başardık. Daha fazla ortak kültür model sistem kullanarak, gösterdiğimiz makrofajlar yayımlanan exosomes ifade bronş epitel hücrelerdeki sıkı kavşak proteinlerin bozabilir. Bu sonuçlar 1) exosomal mekik ile doğuştan gelen bağışıklık ve yapısal hücreler arasında çapraz konuşma katkıda bulunmak için inflamatuar yanıt ve yapısal bariyer ve 2) hedefleme bu miRNAs bozulma tedavisi için yeni bir platform sağlamak öneririz ALI ve ARDS.

Septik akciğer yaralanmalara neden fareler mayi LPS (15 mg/kg) ile tedavi edildi. LPS yönetim 24 h içinde nötrofilik akını akciğerlerde, Şekil 1A' gösterildiği gibi görüldü. Yalıtım ve arıtma exosomes ardından fare BAL sıvısı çizilmiştir. BALF exosomes morfoloji elektron transmisyon mikroskopisi (Şekil 1B) tarafından doğrulandı.
Seçici pro-inf...

Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Bronchoalveolar lavaj Exosomes septik akciğer Lipopolysaccharide kaynaklı yaralanmalarında

Fareler için mayi LPS maruz miRNAs ile paketlenmiş exosomes solunum-alveoler lavaj (BAL) sıvı salgılar. Ortak kültür sistemiyle, biz BAL sıvısı serbest exosomes ifade bronş epitel hücrelerdeki sıkı kavşak proteinlerin bozabilir ve akciğer hasarı vurgulamak pro-inflamatuar sitokinlerin ifade artış gösteriyor.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

bronchoalveolar-lavage-exosomes-lipopolysaccharide-induced-septic

Universidad San Sebastian

Research

JoVE Journal

Genetics

10.8K Görüntüleme.  Atlanta VAMC. Fareler için mayi LPS maruz miRNAs ile paketlenmiş exosomes solunum-alveoler lavaj (BAL) sıvı salgılar. Ortak kültür sistemiyle, biz BAL sıvısı serbest exosomes ifade bronş epitel hücrelerdeki sıkı kavşak proteinlerin bozabilir ve akciğer hasarı vurgulamak pro-inflamatuar sitokinlerin ifade artış gösteriyor.

Video: Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Taze izole edilmiş insan Villöz sitotrofoblastlar ilgili siRNA transfeksiyonu ve EMSA analizi

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Genetik

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

Edu Boyama tarafından Drosophila Yetişkin Yumurtalıkların Mitokondriyal DNA replikasyonu Yerinde Etiketlenmesi içinde

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Histon-miniSOG Kullanılan optogenetic rastgele mutagenez<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Yetiştirme ve Gizle böcek RNA aracılı gen demonte çift sarmallı,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Havuza alınan shRNA MeCP2 reaktivasyonu üzerinde etkin olmayan X kromozomu için ekran

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Düzenleyici elemanlarının ölçü Kromatin erişilebilirlik memeli hücrelerinde formaldehit destekli izolasyon

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Kromatin döngü yapısı yeniden yapılanma CRISPR-aracılı

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Fare embriyonik kök hücre kullanarak Southern blot ve polimeraz zincir reaksiyonu Homolog rekombinasyon olaylar tanımlaması

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Histon translasyonel modifikasyon değişiklikler Maya nörodejeneratif Proteinopathy modellerinde karakterize

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

Sitogenetik teknikle Bemisia tabaci yumurta döllenme oranı belirleme

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...