This research was supported in part by the University of Georgia, and a grant (HD071330) from the National Institutes of Health to MMV.

Bu protokol Georgia Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. 1. Hazırlıklar <ol><li> Taze oosit kültür, satın alma ya da için minimum temel ortam (MEM) hazırlanması ve Tablo 1 &#39;de tarif edilen 3 mg / ml sığır serumu albümini (BSA) ile tamamlar. (Sıfıra dara), bir yükleme dengesine bir polistiren şişe yerleştirin. Amacıyla, BSA hariç tüm reaktif maddelerin ilave edin ve 250 g&#39;lık bir toplam ağırlık ile MQ su ile son hacim getir. Ardından, BSA eklemek çözülür ve filtre sterilize izin verir. Not: Açıklanan MEM ortamı,% 5 CO2,% 5 O2 ve% 90 N2 medikal gaz karışımı ile dengeye ve hücre inkübasyon gerektirmektedir. Bununla birlikte, diğer bir araç (örneğin, CZB) atmosferik şartlar altında% 5 CO2 hücre inkübasyon için izin veren kullanılabilir. </li><li> Oosit mikroenjeksiyon için, satın almak ya da M2 orta hazırlayın. </li><li> P hazırlayın5 IU / 100 ul bir konsantrasyona kadar regnant kısrak serum gonadotropini (PMSG). </li><li> Satınalma ve 1 mcM bir çalışma konsantrasyonu siRNA stoklarını sulandırmak. </li></ol> 2. Fare Oosit Toplama <ol><li> 1. GÜN: over folikül gelişimini teşvik ve ön ovulatuar folikül sayısını artırmak, periton oosit toplama 48 saat önce dişi farelere PMSG 5 IU yönetmek için. </li><li> GÜN 3: Yumurta toplama ve kültür için 1 mcg / ml milrinon ile desteklenmiş MEM / BSA 3 ml 35 mm kültür kaplarına kurdu. Bu fosfodiesteraz inhibitörü olarak oosit tutar profaz-ı tutuklamak ve germinal vezikül dökümü (GVBD) engeller. En az 15 dakika boyunca tıbbi gaz karışımı içinde kültür kaplarına dengelenmesi. Not: Milrinon desteklenmiş ortam oosit toplama ve oosit mikro enjeksiyon ve 24 saatlik kültür tutma sonrası dönem boyunca siRNA gereklidir. </li><li> 15 uygulamaları kurulan laboratuvar ile dişi farelerin yumurtalıkları almak ve önceden ısıtılmış olan yeni bir kültür çanak transfer ve MEM / BSA / milrinon dengelenmiş. </li><li> Oosit toplanması için bir stereomikroskopta aşamasında kültür çanak yerleştirin. Yayın cumulus-oosit kompleksleri (COC) &#39;ın elle 27 G iğne ile antral folikülleri delinmesiyle. 1 ml şırınga bağlı 27 G iğne ile, kültür çanak tabanına bir yumurtalık saptamak ve tüm büyük folikül delinme için ikinci bir iğne kullanımı. Her yumurtalığın için tekrarlayın. </li><li> Kompakt kümülüs hücrelerinin 2 veya daha fazla kat çevrili tüm oosit toplamak, standart ağız pipet prosedürleri veya oosit aspirasyonu diğer araçları kullanma. Yeni bir çanak COC en aktarın ve 1 saat boyunca 37 ° C&#39;de inkübe edin. </li><li> Dikkatle 750 ul&#39;lik bir aspirasyon hacmi ayarlanmış bir 1 ml&#39;lik pipet ile tekrar yumuşak pipetleme çevreleyen kümülüs hücreleri çıkarmak. Pipet adım yaklaşık 12 kez tekrarlayınve oositler 5 dakika süre kurtarmak için izin verir. Tamamını veya kümülüs hücrelerinin çoğu ortadan kadar bu şekilde devam edin. Başka bir kültür tabağına soyulmuş oositler aktarın ve 15 dakika boyunca 37 ° C&#39;de dengelenmeye bırakın. </li><li> Hesaba ilgili hedef karşı spesifik siRNA&#39;lar (i), mikroenjeksiyon, spesifik olmayan (kontrol) siRNA&#39;lar, (ii) mikroenjeksiyon, ve (iii) olmayan bir enjekte edilmiş kontrol grubu üç deneysel gruba soyulmuş oositler tahsis için kullanılacak Kültür koşulları. </li></ol> 3. Oosit Mikroenjeksiyon <ol><li> Oosit Mikroenjeksiyon için milrinon (1 ug / ml) ile takviye edilmiş 3 mi M2 medya ile kültür kaplarına ayarlayın. Yıkama ve enjekte edilen oositlerin daha sonra kültür için MEM / BSA / milrinon ihtiva eden kültür kaplarına hazırlayın. Not: M2 ortamı HEPES tamponu içerir ve atmosferik koşullar altında, 15 dakika boyunca 37 ° C&#39;de ön ısıtmaya gerektirir. MEM tarif tıbbi gaz kullanılarak ön ısınma ve dengeleme gerektirirYukarıda d. </li><li> Mikroenjeksiyon sistemi hazırlayın. 1 mcM belirli siRNA çözeltisi 5 ul enjeksiyon iğnesi yükleyin. Mikroenjeksiyon sistemi mikromanipülatörler tutma pipet ve enjeksiyon iğnesi Güvenli ve kalibre hacmi ve basınç Enjeksiyon Ünitesi kurmak. </li><li> 3 cm kültür çanak kapağın iç tarafında M2 medya 200 ul mikro damla yerleştirin ve kurmak ve mikroenjeksiyon sistemi uyumu için bir oosit ekleyin. </li><li> Bir kılavuz olarak oosit kullanarak, holding ve enjeksiyon iğneleri konumlarını ayarlayın. Nazikçe oosit güvenliğini sağlamak ve hücrenin en geniş çaplı enjeksiyon iğnesi konumunu ayarlamak için tutma pipet negatif basınç uygulayın. Her yerde oosit sitoplazmaya siRNA çözeltisi yaklaşık 10 ul mikroenjeksiyon izin ayarları yapın. <img alt="figür 1" src="/files/ftp_upload/53586/53586fig1.jpg" /> Şekil 1. Oosit mikroenjeksiyon kurdu. ( <strong&gt; sırasıyla sol ve sağ, konumlandırılmış tutma pipet ve enjeksiyon iğnesi ile 3 cm kültür çanak kapağın üzerine yerleştirilmiş M2 medya 200 ul mikro-damla, A) Temsilcisi görüntü. (B) Grubu profaz-ı mikroenjeksiyon öncesinde oosit (GV-evresi) gözaltına aldı. <a href="https://www.jove.com/files/ftp_upload/53586/53586fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> </li><li> Stereomikroskopta sahneye kapağı dönün ve taze M2 medya 200 ul mikro damla ekleyin. Ağız pipetle veya alternatif yöntemlerle mikro damlasına kadar oosit bir grup (~ 10) ekleyin, daha sonra mikroenjeksiyon aşamasına mikro damla ile kapağı dönün. <ol><li> Bireysel oosit Microinject geçin. Yavaş yavaş sitoplazmaya enjeksiyon iğne ucu yerleştirin tutan pipet ile tek oosit Güvenli ve siRNA çözümünün enjeksiyon hacmi sınırdışı. Dikkatle iğne ve mo gerimikro-damla altındaki bir pozisyonda enjekte oosit ziyaretinde. Mikro-damla üstünde bir konuma başarısız enjekte oosit taşıyın. </li><li> Her oosit ile mikroenjeksiyon işlemi tekrarlayın. Her seferinde uygun oosit canlılığı, oosit Microinject az sayıda korumak için. Bu süreç mikro damla sıcaklık ve pH değişiklikleri önlemek için birkaç dakikadan fazla sürmüyor emin olun. </li></ol></li><li> Aspire başarıyla oosit enjekte veya dikkatlice stereomicroscope enjekte oosit mikro damla ile kapağı hareket ettirin. Yıkama ve 37 ° C&#39;de dengelenmeye / BSA / milrinon orta MEM, ağız pipetleme ve alternatif metodlar ile enjekte uygulanabilir oosit aktarın. </li><li> Oosit başka bir grup kullanarak, kontrol grubu için non-spesifik siRNA&#39;lar yüklü yeni bir enjeksiyon iğnesi ile mikroenjeksiyon işlemi tekrarlayın. Ayrı bir kültür çanak MEM / BSA / milrinon ve transfer oosit yıkayın. </li><li> Kültür tüm gruplarınTıbbi bir gaz atmosferi altında, 37 ° C&#39;de MEM / BSA / milrinon 24 saat oocytes. Not: &#39;milrinon blok&#39; kültür dönemi verimli hedef mRNA / protein tükenmesi için gereklidir. </li></ol> Mayotik Matürasyon 4. Oosit Kültürü <ol><li> GÜN 4: oosit oluşturduğu her bir deney grubu için 3 ml MEM / BSA ile kurulumu 4 kültür kaplarına (35 mm). Oosit olgunlaşması için kullanımı,% 10 (yüksek kalitede) cenin sığır serumu (FBS) ile birlikte grup başına bir ortam çanak Supplement. Tüm kültür tabakları tıbbi gaz karışımı ile 37 ° C &#39;de dengelenmeye bırakın. </li><li> Sırayla, &#39;milrinon bloğu&#39; dan oosit serbest MEM / BSA içeren yemekler oosit üç defa yıkamak ve daha sonra% 10 FBS ile ortamı içeren olgunlaşma çanak oosit aktarmak için. Kültür, 37 ° C&#39;de 17 saat boyunca tüm oosit gruplan, yeniden başlamasını ve mayoz ilerlemesini sağlarlar. </li><li> 5. GÜN: oosit çapraz ba¤ hazırlanın çözümlertion, aşağıdakileri içeren: (i) PEM tampon maddesi içinde% 4 paraformaldehid (PFA) (100 mM PIPES [pH 6.9], 1 mM MgCI2, 1 mM EGTA)% 0.5 Triton-X 100, ve (ii) PBS 5 ile takviye edilmiş olan yıkama ve oosit engellemek için kullanılacak% FBS. Not: Bu çözümler öncesinde oosit tespitin 37 ° C-ısınma ön gerektirir. </li><li> (17 saat kültürden sonra) oositi tespit için, hızlı bir şekilde 750 ul ihtiva eden ağız pipetleme ya da (4-iyi bir çanak) bireysel çukurlar halinde alternatif yöntemler ile oosit oluşturduğu her bir deney grubu transferi% 4 PFA çözüm, önceden ısıtılmış ve 37 ° C&#39;de inkübe 1 saat ° C. Daha sonra,% 5 FBS içeren önceden ısıtılmış PBS 750 ul, 3 kez (her biri 15 dakika) oositler, her grup yıkayın. </li><li> 4 ° C&#39;de 200 ul PBS /% 5 FBS O / N Blok oositler bağlama spesifik antikor en aza indirir. </li></ol> 5. İmmünofloresan Analizi <ol><li> GÜN 6: Not: Bağışıklık boyaması bir çok-çukurlu plaka ve solüsyonu ° yapılırytes seri ilgili antikor ve yıkama çözümleri içeren sıralı yuvalara aktarılmıştır. Ya 48 ya da 96 oyuklu plakalar kullanılabilir sadece iyi boyutuna göre çözelti hacmini ayarlar. 96 oyuklu plakalar, oyuk başına 200 için ul kullanın. - 3 yıkanmaktadır - İkincil antikor çözeltisi - 3 yıkanmaktadır primer antikor çözeltisi: Oositler aşağıdaki sırayla farklı çözümler aktarılır. Plaka folyo ile kaplı olabilir ışık maruz kalma sınırı. Taze PBS /% 5 FBS hazırlayın ve antikor çözeltiler elde etmek için, bu çözelti (örneğin, tavşan anti-pericentrin (1/1000), fare anti-tubulin (1/1000)). </li><li> Primer antikor solüsyonu (daha az hacim arzu edildiği takdirde veya 100 ul) 200 ul ihtiva eden tek tek oyuklara her bir deney grubu sabit oosit aktarın ve de parafilm ile kaplayın. Uygun antikor özel koşullara göre inkübe (örneğin 1 saat veya 4 ° CO / N 37 ° C). Not: Primer antikor dilution yanı sıra, özel bir inkübasyon süresi ve sıcaklık, kullanılan farklı antikorlar için uygun koşulları belirlemek için test gerektirir. </li><li> Primer antikor ile inkübe edildikten sonra, PBS /% 5 FBS (10-15 dk) oosit üç kez yıkayın. </li><li> Floresan-konjuge sekonder antikor ihtiva eden çözelti içine oosit aktarın (örneğin, farklı dalga boylarında florokromlar ile konjuge edilmiş anti-tavşan ve anti-fare antikorlarının 1/1000 seyrelti). 37 ° C &#39;de 1 saat süreyle inkübe edin. </li><li> (- 15 dakika her biri 10) PBS /% 5 FBS oosit üç kez yıkayın. </li><li> Son yıkama adımından sonra, temiz bir cam slayt üzerine oosit transferi ve hafifçe herhangi bir fazla yıkama solüsyonu aspire. Bu cam yüzey üzerinde oosit hareketsiz olacaktır. Medya (DAPI içeren) Montaj 8 ul ekleyin ve dikkatli bir 22 mm x 22 mm kapak kayma montaj ortamı kaplaması. Oosit, hava kabarcığı ve / veya zarar vermemek için yavaş yavaş lamel indirin. Not: Alternatif olarak, konfokal mikroskopi analizi için oositin 3 boyutlu özelliklerini korumak önceki slaytta montajı için kapak kayma köşelerine 100 mikron cam boncuklar (vazelin ile karışık) küçük hacim eklemek için. </li><li> 4 ° C&#39;de saklayın saydam ya öz değişmesine ait ilerlemesi değerlendirilmesi olarak kullanılan sekonder antikor eşleştirmek için gerekli olan filtreler ile donatılmış bir fluoresans mikroskopu kullanılarak ifade seviyeleri ve subsellüler protein lokalizasyonu analizine devam edin. </li></ol>

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The authors have nothing to disclose

Örneğin elektroporasyon transfeksiyon ve somatik hücreler, ekzojen nükleik asit aktarımı için çok sayıda yöntem vardır birlikte, mikro-enjeksiyon, transkripsiyonel olarak hareketsiz bir fare oositlerine RNA moleküllerinin verilmesi için uygun bir yöntemdir. Mevcut protokol farklı mili ve / veya oositlerde MTOC ilişkili faktörler fonksiyonel test sağlayan spesifik mRNA in vitro tükenmesi için etkili bir yaklaşım sağlar. Bu yaklaşım, verimli transkript tükenmesi ile sonuçlanır ve son d...

Mayoz gametlerin (oosit ve sperm) oluşur ve mayoz-I ve mayoz-II, sırasıyla 1 sırasında homolog kromozom ve kardeş kromatidler ayırmak için DNA sentezini müdahale olmadan iki ardışık bölümler içeren benzersiz bir bölünme sürecidir. Oositlerde öz değişmesine ait bölünme sırasında kromozomların dağılması hatalar fertilizasyon esnasında embriyo tarafından devralınır anoploidi, neden olabilir. Özellikle, gelişmekte olan embriyolarda anöploidi sıklığı anne yaşı ilerledikçe artar ve kadınlarda 1,2 gebelik kaybı yanı sıra konjenital doğum kusurları önemli bir nedenidir, bu nedenle, mayoz bölünme sırasında anöploidinin moleküler temelini anlamak için önemli bir ihtiyacı vurgulayan . Hücre bölünmesi sırasında, kromozom ayrımı ters göbeğine doğru eki için mikrotübül mili aparatı ve istikrarlı kromozom-mikrotübül etkileşimlerin kurulması montajı ile ilgili hayati bağlıdırkutuplar. Önemli olarak, memeli oositlerinde öz değişmesine ait iğ formasyonu somatik hücrelerde mitoz farklıdır ve benzersiz mikrotübül organize merkezleri sentriyoller 3,4 eksikliği (MTOCs) tarafından düzenlenir. Mikrotübül çekirdeklenme ve organizasyon için gerekli esansiyel proteinler mikrotübül düzeneğini katalize γ-tubulin dahil oosit MTOCs, lokalize. Buna ek olarak, MTOCs 5 de bağlanan bir esas iskele proteini olarak işlev görür ve ankraj γ-tubulin gibi diğer faktörler pericentrin. Özellikle, çalışmalarımız önemli MTOC ilişkili proteinlerin tükenmesi mayoz iğ organizasyon bozar ve tam mili montaj kontrol noktasında (SAC) 6,7 tarafından çözüldü olmayan oosit kromozom ayrımı hataları neden olduğunu göstermektedir. Bu nedenle, mayoz tutuklanmasını tetiklemez iğ istikrar kusurları, anöploidi katkıda önemli bir risk oluşturmaktadır. Mili montaj ve organizasyon, oosit MTOC prot önemli rolleri rağmenein kompozisyon ve fonksiyon pek iyi anlaşılamamıştır. Hücreler kısa mayoz 8,9 yeniden başlaması öncesinde transkripsiyonel sakin olmak gibi memeli oositlerinde belirli hedef proteinlerin fonksiyonlarını test zordur. Bu nedenle, ön-ovulatuar oosit mayoz devam etmek için anne mRNA mağazaları güveniyor ve mayoz bölünme yanı sıra gübreleme 10,11 sonra ilk bölünme bölünmeleri destekler. Memeli oositlerinde mRNA transkriptlerinin RNA interferans (RNAi) aracılı degradasyon etkinliği iyi bilinmektedir ve mayoz olgunlaşma sırasında çeviri için işe anne RNA&#39;lar siRNA 12-14 hedefleme için özellikle uygundurlar. Bu nedenle, oosit içine kısa müdahale RNA&#39;lar mikroenjeksiyon (siRNA) fonksiyonel test için hedef mRNA&#39;ları tüketmek için değerli bir yaklaşım sağlar. Burada, fare oositleri ve spesifik Transcri siRNA aracılı tükenmesi izolasyonu için yöntemleri tarif ederPTS pericentrin temel bir MTOC ilişkili proteinin fonksiyonunu test etmek. Buna ek olarak, oositlerde öz değişmesine ait iğ formasyonunun değerlendirmek için immünofloresan analiz koşulları açıklar.

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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pregnant Mare&#39;s Serum Gonadotropin (PMSG)</td>
			<td>EMD Biosciences</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimal Essential Medium (MEM)</td>
			<td></td>
			<td></td>
			<td>*Recipe outlined in Table 1</td>
		</tr>
		<tr>
			<td>Earle&#39;s Balanced Salt Solution (10x)</td>
			<td>Sigma</td>
			<td>E-7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S-5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvic Acid, sodium salt&#160;</td>
			<td>Sigma</td>
			<td>P-5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G, potassium salt&#160;</td>
			<td>Sigma</td>
			<td>P-7794</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate&#160;</td>
			<td>Sigma</td>
			<td>S-9137</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Sigma</td>
			<td>G-8540</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, disodium salt dihydrate&#160;</td>
			<td>Sigma</td>
			<td>E-4884</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential Amino Acids (50x)</td>
			<td>Gibco&#160;</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamin Mixture (100x)</td>
			<td>Sigma</td>
			<td>M-6895</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red solution</td>
			<td>Sigma</td>
			<td>P-0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td></td>
		</tr>
		<tr>
			<td>Milrinone</td>
			<td>Sigma</td>
			<td>M4659</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.01</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbryoMax M2 Media with Hepes</td>
			<td>EMD Millipore</td>
			<td>MR-015-D</td>
			<td></td>
		</tr>
		<tr>
			<td>siRNAs targeting Pericentrin</td>
			<td>Qiagen</td>
			<td>GS18541</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative control siRNAs&#160;</td>
			<td>Qiagen</td>
			<td>SI03650318</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Hyclone</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Pericentrin (rabbit)</td>
			<td>Covance</td>
			<td>PRB-432C</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetylated a-tubulin (mouse)</td>
			<td>Sigma</td>
			<td>T-6793</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-21430</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti -mouse Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Major Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope (SMZ 800)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Upright Fluorescent Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet Micro-injections System</td>
			<td>Eppenforf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro manipulators</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-injection needles (femtotips)</td>
			<td>Eppendorf</td>
			<td>930000035</td>
			<td></td>
		</tr>
		<tr>
			<td>Holding pipettes (VacuTip)</td>
			<td>Eppendorf</td>
			<td>930001015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>35mm culture dishes</td>
			<td>Corning Life Sciences</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3367</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 mm CA Filter System</td>
			<td>Corning Life Sciences</td>
			<td>430768</td>
			<td></td>
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</table>

meiotic spindle assessment in mouse oocytes by sirna-mediated silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

SiRNA&#39;lar Mikroenjeksiyon in vitro farklı hedef faktörlerinin etkin ve yüksek düzeyde spesifik fonksiyonel test sağlar oositlerde mRNA bozulması ve daha sonra da protein tükenmesi için etkili bir yaklaşım sağlar. Daha sonra, belirli bir fenotip immünofloresan analizi için kullanılan olarak siRNA enjekte edilmiş oositlerde protein tükenmesi doğrulamak için. Mevcut örnekte, anti-tubulin ve anti-pericentrin antikorları ile birlikte DAPI ile ayrı oosit floresan etiketleme etkin: olarak peri...

Watch this Scientific Journal Video about Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing at JoVE.com

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Burada, fare oosit mayoz mili montaj ve organizasyon değerlendirmek için immünoflüoresans analizi ile takip özgü siRNA aracılı mRNA tükenmesi için bir protokol mevcut. Bu protokol, transkript in vitro tükenmesi ve farklı mili ve / veya oositlerde MTOC ilişkili faktörler fonksiyonel değerlendirmesi için uygundur.

SiRNA aracılı susturma Mouse Oositlerinin içinde mayoz Mili Değerlendirmesi

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

meiotic-spindle-assessment-mouse-oocytes-sirna-mediated

Research

JoVE Journal

Biology

11.6K Görüntüleme.  College of Veterinary Medicine, University of Georgia.  Burada, fare oosit mayoz mili montaj ve organizasyon değerlendirmek için immünoflüoresans analizi ile takip özgü siRNA aracılı mRNA tükenmesi için bir protokol mevcut. Bu protokol, transkript in vitro tükenmesi ve farklı mili ve / veya oositlerde MTOC ilişkili faktörler fonksiyonel değerlendirmesi için uygundur. 

Video: Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Fare Oosit içine Nükleer Transfer

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Biyoloji

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

Fare Oosit alımı

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Hamster Oosit ve Fare Embriyolar Toplama ve Kriyoprezervasyon

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

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 ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

İyon Kanalları incelenmesi için yama Kelepçe ve Perfüzyon Teknikleri cinsinden ifade edilir Xenopus Oosit

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Fare Mayotik Hücreler Sitometrisi Arıtma

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Canlı Hücre Kalsiyum Görüntüleme siRNA aracılığı ile gen susturma ile birlikte Ca tanımlar 2 + ER Membran Kaçak Kanallar ve Düzenleme Mekanizmaları

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Pamuk olarak Agrobacterium Virüs Bağlı Gen susturma Testi

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Katyonik lipozomlar üzeri özel siRNA tekrar teslim ile Fare Akciğerler Intersectin-1s uzun süreli suskunluğu. Elektron Mikroskobu ile demonte Etkilerinin Değerlendirilmesi

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...