Biz yazının yararlı yorumlar için temsili sonuçlar Şekil 3 ve Jason Watts ve Chris Webster gösterilen ön deneyler için Chris Webster teşekkür ederim. Bu çalışma için fon JLW için Ulusal Sağlık Enstitüleri (ABD) (R01DK074114) bir hibe sağlanmıştır. Bu çalışmada kullanılan bazı nematod ırkları Araştırma Altyapı Programları NIH Ofisi (P40 OD010440) tarafından finanse edilmektedir Caenorhabditis Genetik Merkezi tarafından sağlandı.

Çoklu doymamış yağ asitleri, ısı, ışık ve oksijen duyarlıdır. Yağ asidi takviyesi tabak hazırlarken Bu nedenle, bakım alınmalıdır yağ asitleri aşırı ısı ve ışığa maruz kalmayacakları şekilde. % 0.1 Tergitol (NP-40) ihtiva eden NGM ortamı yağlı asit sodyum tuzları, sabit karıştırma ile ilave edilir ve bundan sonra, otoklav ve kısmen soğutulur. Plakalar karanlıkta kurumaya bırakılır. C. tarafından yağ asitlerinin Uptake daha sonra gaz kromatografisi ile takip edilebilir elegans, bu plakalar üzerinde kültürlendi. 1.. Yağ asit vasıta hazırlanması <ol><li> Uygun büyüklükte bir balona medya bileşenleri ölçün. 1 litre başına, 17 g Bacto-agar, 2.5 g tripton, 3 g NaCl, etanol içinde eritildi, 1 ml 5 mg / ml kolesterol ve su içinde eritildi, 10 ml% 10 Tergitol ekleyin. </li><li> Millipore su istenen son hacminin% 80 ekleyin ve farklı sayısına eşittir (boş cam şişe ile birlikte ortam otoklavyağlı asit test edilecek konsantrasyonlar), bunun yanısıra, uygun dereceli silindirler. </li><li> 55 ° C&#39;ye ayarlanmış bir su banyosu içinde Serin medya Ortam, cam şişe açık kırılma güvenlik önlemleri ile ve cam parçacıkları yağ asitleri ile karışmaz sağlanarak, yağlı asit sodyum tuzunun çalışma stok çözeltisi hazırlamak soğurken. 100 mM çalışma stok yapmak için yeterli yağ asidi dışarı tartılır. </li><li> , Saflaştırılmış su ile 100 mM&#39;lik bir son konsantrasyon elde etmek için yağlı asit çözeltisi getirin. Tam yağlı asit sodyum tuzunu çözerek, tipik olarak yaklaşık 20-30 dakika sürer. Bir atıl gaz ile temas oksidasyonunu engeller, çünkü, argon ya da azot ile birlikte bir yağ asidinin çalışma stok temizleyin. Kapatılır ve ortam soğuyana kadar karanlıkta çalışma stok saklayın. </li><li> (İsteğe bağlı) RNAi ortam istendiği takdirde, ampisilin ölçmek ve burada izopropil β-D-1-tiogalaktopiranosid (IPTG) çözümleri. </li><li> Agar 55 ° soğuduğunda 1 L başına eklemek C: 11 M MgSO 4 ml 1 M CaCI2 1 ml ve fosfat tamponu 25 ml (1 L başına 108.3 g KH 2 PO 4 ve 35.6 g K 2 HPO 4 otoklav). Filtre sterilize ampisilin ve IPTG çözümler RNAi yapım plakaları ise ekleyin. Plakalarının her türlü için, 1 L&#39;lik nihai ortam hacmi getirmek için steril su ekle </li><li> Bir alev yakınında, Otoklavlanmış aktarmak medya ve uygun Mezür boyutta ve daha sonra istenen hacme ılık steril su ekleyin. Geri ilk şişeye aktarmak medya ve karıştırarak karıştırın. </li><li> Bir alev civarında, bir otoklav ile ölçülerek test edilecek konsantrasyonlarının sayısına eşit bir dizi şişe içine transferi ortam ve uygun bir şekilde dereceli silindir boyuttadır. Yağlı asit çalışma stok tamamen çözülene kadar bir su banyosu kullanılarak bir sıvı olarak aliquoted ortamı koruyun. </li><li> Medya dokunmak sıcak, sıcak ama değil kadar bir heyecan plaka ve karıştırın üzerine bir şişe yerleştirin. Yeterince kendinizi bırakın emin olunyağlı asit stok çözeltisi içinde ilave edin ve ortam katılaşmaya başlamadan önce plakalar dökmek süresi. Heyecan plaka sıcaklığı denetimi varsa, 55 ° C&#39;ye ayarlayın </li><li> İstenen nihai konsantrasyon için ortama 100 mM yağlı asit çalışma stokları seyreltin. Stir ortam beyaz çökelti kadar çözelti (yaklaşık 1 dakika) bulunmaktadır. Medya hala sonra biraz bulanık olabilir. </li><li> (İsteğe bağlı) RNAi ortam 2 mM nihai bir konsantrasyona kadar IPTG ml ve 0.1 mg / eklenti ampisilin isteniyorsa. </li><li> 8 60 mm tabak başına ortam ml veya 100 mm plaka başına ortam 25 ml ilave otomatik bir pipet yardımı ile steril bir 25 ml bir pipet kullanılarak ortam dökün. Kalan şişeler için adımlar dökme yağlı asit ilaveli ve plaka tekrar edin. </li><li> Agar katılaşmış sonra, hafif oksidasyondan korumak için, iyi havalandırılan bir çekmece içerisinde oda sıcaklığında bir kutu ya da mağaza ile yağ asidi-takviye plakaları kapsamaktadır. </li><li> Tohum E. coli OP50 plakaları kurutuldu olan iki gün sonra, plakalar üzerine.60 mm tabak için, (37 ° C&#39;de yetiştirilen), bir gece boyunca OP50 kültür pipet 300 ul. RNAi plakalar boşaltılır olsaydı, plakalar dört gün boyunca kurutulduktan sonra, RNAi bakteri 300 ul tohum. Levha kuruma süresi nedeniyle çeşitli laboratuar ortamında nem farklılıklarından ayarlanması gerekebilir. </li><li> Bakteriyel çim kururken, oda sıcaklığında karanlık ortamda inkübe edin. </li></ol> 2. DGLA&#39;nın Suplementasyon tarafından Germ Hücre Destruction Inducing <ol><li> C. 0.3 mM DGLA (20:03 n-6) içeren plakalar üzerinde büyüdü elegans nedeniyle hem larva ve ergin nematod germ hücrelerinin imha steril hale gelir. <ol><li> Alkali hipoklorit çözeltisi ile muamele edilerek gebe çift cinsiyetli L1 larvalarının bir senkronize bir nüfus hazırlayın (10 ml solüsyon için: 5M NaOH, çamaşır suyu 2.5 ml, 7 ml H2O içinde 0.5 ml) eklenmiştir. Yavaşça yetişkin kurt kadar bu çözelti içinde gebe çift cinsiyetli kayas çözülür. Yumurta korunur ve düşük hızlı santrifüjleme ile tane haline edilebilir. </li><li> (3 g KH 2 PO 4, 6 g HPO 4, 5 g NaCl, 1 ml 1 M MgSO 4 H, 2 O L. otoklav sonrası MgSO 4 1 ekleyin 2 Na) M9 tampon maddesi içinde yumurta terkip 3 kez yıkayın. Yeterli oksijen sağlamak için, bir gece boyunca bir çalkalayıcı üzerinde M9 tampon ve kaya 5 ml&#39;lik bir son hacim için 15 ml plastik bir tüp içinde yumurta tekrar süspansiyon. </li><li> L1 larva iki gün RNAi bakteri sonra kaplama OP50, ya da bir gün ile tohumlamadan sonra takviye edilmiş plakalara ilave edilmelidir. Onlar yetişkin aşamasına ulaşana kadar üç gün boyunca 20 ° C&#39;de inkübe solucanlar ya. </li></ol></li><li> Yetişkin solucanlar rahim içinde gelişmekte olan embriyolar görselleştirmek için yeterince yüksek büyütme ile bir diseksiyon mikroskobu kullanarak sterilite için attı olabilir. Başarılı germ hücre kaybı yumurta açık bir rahim boşluğu olarak görünecektir. </li><li> Solucanlar da sabitleme ve nükleik a ile boyanarak attı olabilirgerm hücreleri çekirdeklerin görselleştirme ve gelişen embriyolar kolaylaştırmaktadır cid boya diamindinophenylindole (DAPI). Hızlı bir DAPI leke bir gözlem 9 M9 tampon bir damla halinde solucan toplama ile elde edilir. <ol><li> , 0.2 ng / m 1 ml Sel-cam. DAPI% 95 etanol çözeltisi içinde, yaklaşık 5 dakika boyunca bekletin. </li><li> Bir slaytta Vectashield bir damla içine solucanlar almak ve daha sonra bir lamel ile kaplayın. </li><li> Optimum boyama için karanlık gecede 4 ° C&#39;de mühür lamel ve mağaza, ancak slaytlar de hemen görülebilir. Varlığında veya bir UV lambası ve filtre ile teçhiz edilmiş bir floresans mikroskobu kullanılarak germ hücre çekirdeklerinin olmaması için puan. </li></ol></li></ol> 3. Gaz Kromatografisi ile Yağ Asit Alımı Onaylayan C. Genel olarak yağ asidi bileşimi, elegans daha sonra ayrılır ve gaz Chr kullanılarak nicelendirilir yağlı asit metil esterleri (FAME) üretilmesi ile belirlenebiliromatography 4. <ol><li> Su ile plakalar kapalı yıkamadan ve silanize edilmiş 13 mm x 100 mm cam tüpe Üstü yivli aktarılarak 500-1.000 erişkin solucanlar toplayın. </li><li> Solucanlar yerçekimi tarafından razı olsun, ve sonra bir cam Pasteur pipeti ile mümkün olduğunca çok su çıkarmak. </li><li> Su ile bir kez solucanlar yıkayın ve daha sonra tekrar mümkün olduğu kadar çok suyu çıkarmak. </li><li> Fames bir su banyosu içinde 1 saat boyunca 70 ° C&#39;de% 2.5 1 H 2 ml metanol içinde SO 4 ve daha sonra ısıtma eklenerek oluşturulur. </li><li> Su banyosundan çıkarın ve tüpler, 1 dakika için soğutun. </li><li> 1.5 ml su ve 0.25 ml heksan eklenerek Fames ekstrakte edin. </li><li> Recap tüpler ve şiddetle çalkalanır. </li><li> , Sulu çözücüden ayrılması için heksan maksimum hızda 1 dakika boyunca masa üstü bir klinik santrifüj cihazında santrifüj tüpleri. </li><li> Sulu fazın bir transferi için dikkatli olmak, bir GC şişe içinde bir GC şişe içeriğine heksan (üst katman) aktarın. GC için analysis, heksan içinde FAME 1-2 ul kareleri analizi için uygun olan bir polar kılcal gaz kromatografi sütunu üzerine enjekte edilir. Agilent 7890 GC enjektör 1.4 ml / dk &#39;lık bir akış oranı ile, 250 ° C&#39;de ayarlanır ve GC fırın 1 dakika için tutulan 130 ° C&#39;lik bir başlangıç ​​sıcaklığı için programlanır. Bundan sonra, sıcaklık 10 ° C / dk kadar 190 ° C rampalı edilir ve daha sonra 210 ° C&#39;ye kadar 5 ° C / dk &#39;da yeniden rampalı ve ek 1 dakika tutuldu. </li><li> , DGLA&#39;nın bu uptake oluştu sağlamak C tanımlanması için otantik standartlar kullanılarak alev iyonlaşma algılaması (FID) ya da kütle spektrofotometresi (MS) tespiti ile Fames analiz etmek yağ asitleri elegans. </li></ol>

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Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Burada C takviyesi için bir yöntem tarif Diyet doymamış yağ asitleri ile elegans. Yukarıda belirtildiği gibi, PUFAların en çift bağların reaktif doğası, bu yağ asitleri, ısı ve ışık 11 ile oksidasyona karşı duyarlı olması neden olur, çünkü bakım PUFA takviye plakaları hazırlanmasında alınmalıdır. Oksidasyonu önlemek için, ortam karanlık bir ortamda 55 ° C ve depolar plakalara soğuduktan sonra, sıvı agar ortamına PUFA eklemek için önemlidir. <p c...

Yağ asitleri temel yapı membran bileşenleri hem de verimli enerji depolama moleküllerdir. Ek olarak, yağ asitleri, lipazlar tarafından hücre zarlarından parçalanabilen ve enzimatik olarak işaret efektör 1 üretilmesi için değiştirilebilir. Doğal olarak oluşan çok-doymamış yağlı asitleri (PUFAlar) iki veya daha fazla cis çift bağ ihtiva edebilir. Omega-3 yağ asitleri ve omega-6 yağ asitleri, yağ asidi metil sonuna göre çift bağların pozisyonlara göre birbirinden ayırt edilmektedir. Sağlıklı beslenme omega-6 ve omega-3 yağ asitleri hem de gerektirir. Ancak, Western diyetler omega-6 yağ asitleri ve omega-3 yağ asitleri zayıf özellikle zengindir. Yüksek bir omega-6 omega-3 yağ asidi oranı, kardiyovasküler ve enflamatuar hastalıkların riski Bununla birlikte, belirli yağ asitlerinin tam yararlı ve zararlı işlevleri de 2 anlaşılmamıştır ile ilişkilidir. Yuvarlak kurt Caenorhabditis elegabir omega-3 desatürazı, çoğu hayvan 3,4 in mevcut olan bir aktivitesi omega-6 ve omega-3 yağ asitleri, bir dizi üretmek için gerekli olan bütün enzimleri sentezler için ns yağlı asit fonksiyonu çalışmalarında yararlıdır. Yağlı asit desatüraz enzimleri eksik olan mutantlar, gelişim ve nörolojik kusurlar 4-6 bir dizi yol açan, özel PUFAların üretilmesi için başarısız. Diyet yağ asitlerinin fizyolojik etkilerini incelemek için, C de mutant ve RNAi yok etme teknikleri kullanarak genetik analizi ile uyumlu bir biyokimyasal analiz geliştirdik elegans. Özel PUFAlar ile takviyesi dökülmesinden önce agar ortamına bir yağlı asit sodyum tuzu çözeltisinin eklenmesi ile elde edilir. Bu, E. tarafından PUFA alımı ile sonuçlanan Bu bakteri membran birikir coli besin kaynağı. C. elegans PUFA içeren bakteri yemek ve bu diyet takviyesi arızalı kurtarmak için yeterlidirPUFA eksikliği mutantlarının ts. En çok yağlı asitlerin takviyesi, ancak, belirli bir omega-6 yağ asitleri, özellikle dihomo-gama linolenik asit (DGLA, 20:03 n-6) C arasında kalıcı bir imha neden yabani tip hayvanlar üzerinde herhangi bir zararlı etkisi yoktur elegans germ hücreleri 7,8. Gaz kromatografisi bakteriyel besin kaynağı (OP50 veya HT115 olarak) hem de nematod takviye yağ asidi alımını izlemek için kullanılır. Medyada deterjan Tergitol (NP-40) &#39;nin ilave edilmesi, tüm plaka ve E ile yağlı asitlerin daha etkin alınması yoluyla yağlı asitlerin dağılımını sağlar: coli ve nematodlar. Biz, doymamış yağ asitleri kolayca bakteri ve C. tarafından alınır bulduk elegans, fakat doymuş yağlı asitlerin alımını çok daha az etkilidir. Bu makalede, yağ asitleri ile agar ortamı tamamlamak için nasıl adım adım tarif, hem de inci yağ asidi alımını izlemek için ne kadar olacakgaz kromatografisi kullanılarak e nematod.

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			<td height="22" style="height:22px;width:208px;">Bacto-Agar</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">214010</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tryptone</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">211705</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">NaCl</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3624-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tergitol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">NP40S-500mL</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Cholesterol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">C8667-25G</td>
			<td style="width:275px;">(5 mg/mL in ethanol)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">MgSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">2504-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">CaCl2</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">1311-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">K2HPO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3254-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">KH2PO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3246-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sodium dihomogamma linolenate</td>
			<td style="width:128px;">NuCHEK</td>
			<td style="width:155px;">S-1143</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Warm sterile Millipore water</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile water for collecting worms</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Nuclease-free Water for DGLA stock solution</td>
			<td style="width:128px;">Ambion</td>
			<td style="width:155px;">AM9932</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Ampicillin</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">BP1760-25</td>
			<td style="width:275px;">100 mg/ml in water (for RNAi plates)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:128px;">Gold Biotechnology</td>
			<td style="width:155px;">12481C100</td>
			<td style="width:275px;">1 M in water (for RNAi plates)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">HSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">9681-03</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Methanol</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">A452-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Hexane</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">H302-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">diamindinophenylindole (DAPI)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">D9542</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">VectaShield</td>
			<td style="width:128px;">Vector Laboratories</td>
			<td style="width:155px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Glass Flask</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass bottles with stirbars</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">FB-800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass Graduated Cylinder</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">08-557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Stir Plate</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">97042-642</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Waterbath at 55+ &#176;C</td>
			<td style="width:128px;">Precision Scientific Inc.</td>
			<td style="width:155px;">66551</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Screwcap Brown Glass Vial</td>
			<td style="width:128px;">Sun SRI</td>
			<td style="width:155px;">200 494</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Argon gas tank</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Automated Pipette aid</td>
			<td style="width:128px;">Pipette-Aid</td>
			<td style="width:155px;">P-90297</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile Serological Pipettes (25 ml)</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4489</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bunsen Burner</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">89038-534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Dissection microscope</td>
			<td style="width:128px;">Leica</td>
			<td style="width:155px;">TLB3000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Silanized glass tube</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:155px;">STT-13100-S</td>
			<td>for FAMEs derivitization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PTFE Screw caps</td>
			<td style="width:128px;">Kimble-Chase</td>
			<td style="width:155px;">1493015D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Clinical tabletop centrifuge</td>
			<td style="width:128px;">IEC</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Crimp Vial</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial Insert</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 232</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial cap</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Gas Chromatograph</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">7890A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Mass Spectrometry Detector</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">5975C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Column for gas chromatography</td>
			<td style="width:128px;">Suppelco</td>
			<td style="width:155px;">SP 2380</td>
			<td>30 m x 0.25 mm fused silica capillary column</td>
		</tr>
	</tbody>
</table>

dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans

Yağ asitleri çok sayıda hücre fonksiyonları için gereklidir. Bu membran hidrofobik özünü oluşturan ve çeşitli sinyal yoluna katılabilir, verimli enerji depolama molekülleri karşılık vermektedir. Caenorhabditis elegans omega-6 ve omega-3 yağ asitleri, bir dizi üretmek için gerekli olan bütün enzimleri sentezler. Bu, basit anatomi ve mevcut genetik araçları yelpazesi ile birlikte, bu yağ asidi işlevini incelemek için çekici bir model olun. Diyet yağ asitlerinin fizyolojik etkilere aracılık genetik yollarının araştırılması amacıyla, C takviyesi için bir yöntem geliştirdik Doymamış yağ asitleri ile elegans beslenme. Takviyesi solucanlar yağ asidi bileşimini değiştirmek için etkili bir araç ve aynı zamanda yağlı asit eksikliği olan mutantlar kusurları kurtarmak için kullanılabilir. Önerilen yöntem, yağ acidsodium tuzları ile takviye edilmiş nematod büyüme ortamı, agar (NGM) kullanır. Takviye edilmiş plakalarda yağ asitleri incorpora olmakted sonra C. tarafından alınır, bakteriyel besin kaynağı, zarların içine takviye bakteriler üzerine yem elegans. Biz de desteklenmiş solucanlar meydana yağ asidi kompozisyonundaki değişiklikleri izlemek için bir gaz kromatografisi protokol açıklar. Bu C. büyük ve küçük popülasyonların diyetlerine ek etkili bir yoldur elegans, bu yöntem için bir dizi uygulama için izin verir.

C. takviyesi elegans beslenme bakteriyel zarı içine yağ asidi alımı ve birleştirmek için bakteriyel gıda kaynağının kabiliyeti ile sınırlıdır. E. yeteneğini belirlemek için bu membran içine çeşitli yağ asitleri asimile coli OP50, OP50 hiçbir takviyesi, 0.1 mM ve stearik asit (18:00), sodyum oleat (18:1 n-9), ve sodyum DGLA (20, 0.3 mM konsantrasyonları ile ortamı üzerine kaplanmıştır : 3n-6). Tabaklar karanlıkta 2 gün boyunca oda sıcaklığında kurutuld...

Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

&lt;em&gt; Caenorhabditis elegan &lt;/ em&gt; geliştirme ve fizyolojisi çok-doymamış yağlı asitlerin fonksiyonlarını keşfetmek için uygun bir modeldir. Bu protokol &lt;em&gt; C tamamlayan etkili bir yöntem tarif elegans &lt;/ em&gt; çoklu doymamış yağ asitleri ile diyet.

Çoklu doymamış yağ asitlerinin Diyet takviyesi<em&gt; Caenorhabditis elegans</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

dietary-supplementation-polyunsaturated-fatty-acids-caenorhabditis

Research

JoVE Journal

Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

17.0K Görüntüleme.  Washington State University.  Caenorhabditis elegan geliştirme ve fizyolojisi çok-doymamış yağlı asitlerin fonksiyonlarını keşfetmek için uygun bir modeldir. Bu protokol C tamamlayan etkili bir yöntem tarif elegans çoklu doymamış yağ asitleri ile diyet. 

Video: Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

İzolasyon ve In vitro Etkinleştirme Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Biyoloji

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Katı Plaka tabanlı Diyet Kısıtlama Caenorhabditis elegans</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Erken Embriyo Oluşumu Zaman atlamalı Mikroskopi Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Temel<em&gt; Caenorhabditis elegans</em&gt; Yöntem: Senkronizasyon ve Gözlem

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Prostaglandin Ekstraksiyon ve Analiz<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

yetiştirme<em&gt; Caenorhabditis elegans</em&gt; Laboratuvarı Üç boyutlu

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Oksijen tüketimi oranlarında sağlam Caenorhabditis elegans ölçümü

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Arınma ve Caenorhabditis elegans Ekstrasellüler Veziküllerin Analizi

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Caenorhabditis eleganlarında Dokuya Özgü Proteostatik Düşüşün Ölçülmesi

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Caenorhabditis elegans'ta Agrega Niceliğinin Otomatikleştirilmesi

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...