<p class="jove_content"&gt; Biz yazının eleştirel okuma Dr Shuang Liu teşekkür ederim. PCL12 yapı bir hediye formu Dr Christopher D. Bağlantı oldu. Bu çalışma iki Ulusal Bilim Vakfı hibe (0842708 ve 1026958) ve FS için AHA hibe (09GRNT2250529) tarafından desteklenmiştir.</p

1. Transgenik Worms İnşaatı <ol><li> Dönüşüm. <ol><li> Enjeksiyon pedleri hazırlayın. Bir cam lamel üzerine suda çözünmüş sıcak,% 2 agaroz, bir damla yerleştirin. Hızla açılan ikinci bir lamel yerleştirin ve hafifçe dokunun. Agaroz ayrı slayt lamelleri katılaşmış ve 80 ° C&#39;de bir vakum fırın içinde lamel ped ° CO / N fırında sonra </li><li> Pipetler çekin. Biz 1/0.5 mm filament ile OD / ID borosilikat kılcal çekmek için bir Sutter P-97 çektirme kullanın. Pipetler daha sonraki bir aşamada açık kırık kapalı ucu ile dövme. </li><li> Enjeksiyon karışımı hazırlayın. Ul / 150 ng arasında nihai bir konsantrasyona kadar, ilgili DNA (yapı başına 20 ng / ml) artı boş plazmid DNA içeren enjeksiyon karışımı olun. Herhangi bir kirletici kaldırmak için enjeksiyon karışımı santrifüj. Kirletici dönüşüm verimliliği azaltmak için bu adım çok önemlidir. B yeni bir tüp en iyi 5 ul (enkaz ve diğer kirleticiler alt toplamak) transferE enjeksiyon kullanılır. </li><li> Kılcallık tarafından enjeksiyon karışımı yükleyin. Pipet alt enjeksiyon karışımı batırılır. Tipik olarak 0.5 ul 100 solucanlar enjekte etmek için yeterlidir. </li><li> Enjeksiyon pipet yükleyin. Mikromanipülatör sahibinin üzerine pipet yerleştirin ve agaroz pad üzerinde enkaz karşı bir bardak slayt veya alternatif olarak monte edilmiş bir kapak kayma kenarına ucu sürtünme tarafından açık ara. Bu çok büyük ipuçları zarar solucan gibi çok önemli bir adımdır ve çok küçük ipuçları kolay tıkanır. Kırık ucu kalitesi akış hızı en önemlisi şekil ve ile değerlendirilebilir. Akış hızı enjeksiyon basıncına tepki olarak halokarbon 700 bir damla yağ daldırılan açık ucundan akan kabarcıkların büyüklüğü ile değerlendirilebilir. </li><li> Enjeksiyon pad solucanlar aktarın. Bir enjeksiyon pad üzerinde 700 halokarbon petrol bir damla koyun ve yağ (yeni başlayanlar için 1 veya 2) birçok solucanlar aktarın. Kadar ped üzerine solucanlar aşağı itmek için almak bir solucan kullanıny agaroz uygun. Worms aynı yöne bakacak şekilde ventral taraf ile satır odaklı olmalıdır. Solucan kafası dokunmaktan kaçının. Solucanlar ped uymak için başarısız birkaç denemeden sonra ise, yeni bir ped ile değiştirin veya agaroz kalınlığı ve / veya konsantrasyonu artırır. </li><li> Solucan içine pipet takın. Sahne hareket ettirerek, pipet altında solucan yerleştirin. Sitoplazma birçok germ hücre çekirdekleri tarafından paylaşıldığı için gonadın merkezi bir çekirdek pipet yerleştirin. Bu birçok döl enjekte DNA teslim olasılığını artırır. Pipet solucan (~ 15 ° -25 ° açı) neredeyse paralel durmalıdır. Cilt basılana kadar dikey olarak solucan vücudunun aşağı pipet ucu itin. Sonra yavaşça ucu en ekleme ikna etmek için manipülatör dokunun. En iyi sonuç için her iki gonadlar enjekte. </li><li> DNA çözüm enjekte edilir. Gonad şişer kadar pipet basınç uygulayın. Akışını durdurmak ve pipet kapalı solucan çekin. Taşıyın akışı için iğne Test vebir sonraki solucan ve tekrar enjeksiyon adımlar. </li><li> Solucanlar kurtarmak: solucanlar kurtarma tampon bir damla (~ 10 ul) ekleyin ve solucanlar yüzme başlayana kadar kuluçkaya yatmaktadır. Çözüm çok M9 kadar solucan yüzme devam ve birkaç kez tekrar kadar M9 eşit miktarda ekleyin, bekleyin. Bireysel numaralı seribaşı plakaları için solucanlar aktarın. </li></ol></li><li> Entegrasyon. <ol><li> Taze bir tabak içine 40 sağlıklı, iyi beslenmiş, L4 solucanlar yerleştirin </li><li> 40 dakika 4.000 rad ile γ-ışını ile saçmak. Taze, OP50 numaralı seribaşı plakaları (4 solucanlar / plaka) ışınlanmış solucanlar (P0) aktarın. Worms alternatif olarak 300 J / m 2 UV&#39;lerinizi bir doz ile ışınlanmış olabilir. </li><li> Bireysel plakalar her plaka 10-20 F1 transformantlar aktarmak, ilgili P0 kökenli levhalar etiket. </li><li> Ayrı levhalar her F1 için 2-4 F2 transformantlar tek tek. Dönüşüm marker 100% iletimi için F3 döl kontrol edin. Tipik olarak, bir ışınlanmış solucan wil gelen döl% 1-3l bir entegrasyon olay var. </li><li> Üç veya daha fazla bağımsız transformant hatları stokları olun. </li></ol></li></ol> 2. Davranış Tahliller <ol><li> Yaş-senkronizasyon. <ol><li> Standart 10 cm NGM plakalar + OP50 E&#39;de solucanlar büyütün gebe yetişkinlerin büyük bir nüfus kadar coli (3-5 gün) ulaşılır. </li><li> 1 mi M9 tampon bunları süspansiyona, 50 ml Falcon tüpleri solucanları toplayın. </li><li> 3 dakika boyunca 450 x g, 5 ml M9 tampon ve santrifüj ekleyin. Süpernatant atın. 3-4x tekrarlayın. </li><li> Son santrifüj sonunda, süpernatan kaldırmak ve pelet solucanlar temel hipoklorit çözeltisi (0.5 M NaOH ve taze karışık% 1 hipoklorit), 10 hacim ekleyin. ~ 10 dakika boyunca oda sıcaklığında inkübe edin. Lizis Reaksiyon bir lamel üzerine parçalama reaksiyonu bir damla yerleştirme ve bir mikroskop altında incelenmesi ile solucanlar izlenebilir. Solucanların yaklaşık% 80 kırıldığı zaman, reaksiyon durdurulmalıdır. </li><li> Eklenti tarafından parçalama reaksiyonu durdurmaksteril yumurta tampon g aynı hacimde. </li><li> 5 dakika boyunca 450 x g&#39;de santrifüj ile yumurta (ve karkas) toplayın. </li><li> Steril yumurta tampon 2-3x ile yumurta yıkayın. </li><li> M9 tampon ve tohum onları standart NGM plakalar üzerinde yumurta O / N inkübe edin. </li></ol></li><li> Kemotaksis testi. <ol><li> Agar kabaca 0.5x0.5x0.5 cm birkaç adet hazırlayın. Biz 10 cm plaka agar yatırmak ve biz oradan agar parçalar kesti. </li><li> 2 saat boyunca arzu edilen cezbedici (doyurur civarındaki doyurulması konsantrasyonlarda) ihtiva eden bir çözelti içinde, agar parça bekletin. Tipik Cezbedici lisin (0.5 M), biyotin (0.2 M) &#39;dir. </li><li> Bir test spot yerini ve kontrol nokta (Şekil 1A) işaretlenmiş edildiği bir 10 cm testi plaka bir agar yığın Depozito. Dengeleme ve degrade O / N (Şekil 1B) oluşumunu sağlar. Tek bir deneme için 5 tabak hazırlayın. </li><li> Önce deneme 20 mM NaN 3 (Anest 10 ul ekleyinHer noktaya) infographie. </li><li> Plakasının merkezinde 20 yaş senkronize solucanlar yerleştirin. 20 ° C&#39;de inkübatör plaka koyun </li><li> 1 saat sonra, test / kontrol noktaları üzerinde hayvanların sayısı ve aşağıdaki gibi kemotaksis indeksi (CI) hesaplanır: <img alt="Denklem 1" fo:content-width=".863in" fo:src="/files/ftp_upload/50435/50435eq1highres.jpg" src="/files/ftp_upload/50435/50435eq1.jpg" /> N, N-testi ve N Mrk., hayvanların toplam sayısı, test yerinde hayvan sayısı ve kontrol nokta hayvanların sayısını göstermektedir burada. </li></ol></li></ol> 3. Temel embriyonik Hücre Kültürü <ol><li> Yaşa senkronizasyon açıklandığı gibi solucanlar eritilir. </li><li> 5 dakika boyunca 450 x g hızında steril yumurta tampon ve santrifüj ile aynı hacim ekleyerek parçalama reaksiyonu durdurun. Yavaşça pelet yumurta kaybetmemek için dikkatli olmak süpernatant atın. 2-3x tekrarlayın veya süpernatant açıktır kadar. </li><li> Pelet yumurta (ve karkas tekrar süspansiyon) 2 ml steril yumurta tamponu içinde ve yumurta tamponu içinde steril% 60 sukroz, 2 ml ekleyin. Yumurta tamamen elle veya vorteks (onlar santrifüj altında kümeleri oluşturmak eğilimi gibi) yeniden süspanse kadar bu çözüm karıştırın. </li><li> 15 dakika 450 x g&#39;de santrifüj. </li><li> Dikkatli bir şekilde steril tüpe süpernatant (yumurta içeren) aktarın. Karkas ve parçalama yan ürünleri diğer içeren pelet atın. </li><li> Yumurta tamponunda yeniden süspansiyon haline getirilmesini yumurta ve 5 dakika boyunca 450 xg santrifüj kalıntı sukroz çıkarın. Yavaşça toplamak ve süpernatant atın. 3x tekrarlayın. </li><li> Bir laminer başlık altında yumurta kabuğu sindirmek için oda sıcaklığında 1 U / ml kitinaz içeren steril yumurta tampon pelet yumurta tekrar süspansiyon. 30 dakika bu durumda reaksiyon (ters bir hücre kültürü mikroskop altında) izlemek için başlatmak. Çitinaz Her parti, biraz farklı bir aktiviteye sahiptir. Genellikle sindirim 1 saat içinde tamamlanır. </li><li> Yaklaşık% 70-80 yumurta kabuğu kitinaz CM-15 eklenti (L-15 hücre kültürü med sindirilir zamaniyum,% 10 fetal sığır serumu, 50 U / ml penisilin ve 50 ug / ml streptomisin) &#39;tür. Bir 27 göstergesi ile bir şırınga kullanarak hücreleri ayırmak. Sağlam embriyolar kaldırmak için filtre 5.0 mikron, hücre ve larva kümeleri ile hücre süspansiyonu filtre. </li><li> Pelet 450 santrifüjleme ile ayrışmış hücre süspansiyonu, 15 dakika boyunca xg. Süpernatantı ve CM-15 hücre kültürü ortamında pelet tekrar süspansiyon haline getirin. </li><li> . Cam kapak Plaka ayrışmış hücreleri daha önce su Not çözünmüş fıstık lektin (0.1 mg / ml) ile kaplı fişleri: hücreleri ayırt etmek için yüzeye uygun olmalıdır. </li><li> Hücreler, 2 haftadan daha uzun için hava içinde oda sıcaklığına (16-20 ° C) muhafaza edilebilir. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Burada C. kullanarak amylopathies hücresel ve moleküler yönlerini incelemek için, kombine yaklaşım tarif elegans. Bu yaklaşımın avantajları şunlardır:. 1) düşük maliyetli C.elegans oda sıcaklığında, bakterilerle numaralı seribaşı normal bir Petri kabı korunur. 2) Güçlü genetik. Transgenik hayvanlar birkaç ay içinde elde edilen promotör sekansları ve geniş bir dizi spesifik nöronlarda istenen genin ekspresyonunu yürütmek için kullanılabilir edilebilir. 3) Bas...

Beta amiloid (Aβ) β ve γ secretases 1 ile amiloid ön-madde proteininin (APP) sıralı bölünme sonra oluşan 36-43 amino asitlik bir peptiddir. Γ sekretaz Aβ peptidin C-terminal ucu işler ve değişken uzunlukta 5 sorumludur. Aβ en yaygın formları Aβ 40 ve Aβ 42, daha yaygın olarak, AD 5 gibi patolojik durumları ile ilişkili olan ikinci bulunmaktadır. Yüksek konsantrasyonlarda Aβ formu β-yaprak azından amiloid fibriller 6 oluşturmak için agrega. Fibriller yatakları nöronları çevreleyen senil plaklar ana bileşenidir. Plaklar ve yayılabilir, olmayan plak Aβ oligomerler, hem Aβ altında yatan patojenik formları oluşturacağı düşünülmektedir. Nöronal amylopathies Laboratuvar çalışma süresi ile bu bu koşullar ilerleme gerçeği ile karmaşık. Bu nedenle, importanfareler-kısa ömrü için model-tamamlayıcı genetik uysal hayvan geliştirmek için t. Bu modeller amylopathies-genellikle hücresel ve moleküler-ve basitliği sayesinde, sorunun özünü yakalamak için yardımcı belirli yönlerini aydınlatmak için kullanılabilir. Solucan Caenorhabditis elegans düşüyor bu kategorisidir. Bu kısa bir ömrü vardır, ~ 20 gün ve gen ifadesi, protein kaçakçılığı, nöronal bağlantı, sinaptogenez, hücre sinyalizasyon ve ölüm düzenlenmesi de dahil olmak üzere ek temel hücresel süreçleri memeli 7 benzer. Solucanın benzersiz özellikleri güçlü genetik ve bağımsız vasküler hasar nöronal hasarı incelemek için sağlayan bir damar sistemi, eksikliği içerir. Öte yandan, bir beyin eksikliği C kullanımını sınırlamaktadır nörodejenerasyon birçok açıdan eğitim için elegans. Buna ek olarak, lezyonların anatomik dağılımlarının üreme ve kimlik bu organizmada gerçekleştirilemez. Diğer sınırıations gen ekspresyon profilleri ve karmaşık davranış ve hafıza fonksiyon bozukluğu hem de farklılıkları değerlendirmek için zorluk içerir. Burada C oluşturmak için yöntemler tarif amylopathies bir elegans modelleri.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>1. NGM</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>3 g</td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>AMRESCO</td>
			<td>J637</td>
			<td>17 g</td>
		</tr>
		<tr>
			<td>Bacto-peptone</td>
			<td>AMRESCO</td>
			<td>J636</td>
			<td>2.5 g</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 975 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving, then add the following items and mix well</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>C3045</td>
			<td>1 ml of 5 mg/ml stock( in ethanol)</td>
		</tr>
		<tr>
			<td>Potassium phosphate buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>25 ml of 1M stock</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2. Potassium phosphate buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>108.3 g</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td>35.6 g</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>3. M9 buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>3 g</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td>6 g</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4. Egg buffer (pH 7.3, 340 mOsm)</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>118 mM</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>48 mM</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M4880</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5. CM-15</td>
		</tr>
		<tr>
			<td>L-15 culture medium</td>
			<td>Gibco</td>
			<td>11415</td>
			<td>450 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>50 units/ml</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>50 g/ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Adjust to 340 mOsm with sucrose then sterile filter into autoclaved bottles and store at 4 &deg;C</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6. Other Reagents</td>
		</tr>
		<tr>
			<td>Halocarbon 700 oil</td>
			<td>Halocarbon Products</td>
			<td>9002-83-9</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 &mu;m Acrodisc Syringe Filter</td>
			<td>PALL Co.</td>
			<td>4199</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chitinase</td>
			<td>Sigma-Aldrich</td>
			<td>C6137-5UN</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lectin (peanut)</td>
			<td>Sigma-Aldrich</td>
			<td>L0881-10MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L5501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution</td>
			<td>Sigma-Aldrich</td>
			<td>425044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>71289</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a caenorhabditis elegans model system for amylopathy study

Amylopathy zamanla dokularda amiloid beta anormal sentezi ve birikimi (Aβ) tanımlayan bir terimdir. Aβ Alzheimer hastalığı (AH) bir özelliğidir ve Lewy cismi demans, dahil vücut miyozit ve serebral amiloid anjiyopati 1-4 bulunur. Amylopathies giderek zamanla gelişir. Bu nedenle kısa ömürlü basit organizmaların bu koşulların moleküler yönlerini aydınlatmak için yardımcı olabilir. Burada, solucan Caenorhabditis elegans kullanarak Aβ-aracılı nörodejenerasyon incelemek için deneysel protokolleri tarif. Böylece, yetişkin çift cinsiyetli en sinsisyal gonadlar içine kodlayan DNA insan Aβ 42 enjekte edilerek transgenik solucanlar inşa. Transformant çizgiler Mutasyon-kaynaklı entegrasyonu ile stabilize edilir. Nematodlar yumurtalarını toplama ve ekim ile senkronize yaş vardır. Aβ 42 eksprese eden nöronların fonksiyonu (elverişli davranış tahlillerinde test edilir kemotaksi deneyis). Embriyolardan elde edilen primer nöronal kültürler davranışsal verileri tamamlamak için ve anti-apoptotik bileşiklerin nöro-koruyucu etkisini test etmek için kullanılır.

Bizim protokolleri ile, nöronal fonksiyonu 8 insan Aβ 42 oligomer etkilerini incelemek. Bir fragmanını kodlayan insan Aβ 42 ve yangın vektör pPD50.52 yapay olarak sinyal peptidi kodlama sekansı ucunda bir Sma 1 kısıtlama endonükleaz sitesini ortaya primerler kullanılarak yapı PCL12 9 ile amplifiye edildi. Bu kısım daha sonra özel Sma 1 Alanı 10 arasındaki pPD95.75 Acil vektörü içinde bir 2.481-bp FLP-6 promotör sekansı ihtiva eden bir...

Watch this Scientific Journal Video about A Caenorhabditis elegans Model System for Amylopathy Study at JoVE.com

A Caenorhabditis elegans Model System for Amylopathy Study

Biz solucan amylopathies yönlerini incelemek için yöntemler tarif<em&gt; C. elegans</em&gt;. Biz insan Aβ ifade solucanlar oluşturmak için nasıl göstermek<sub&gt; 42</sub&gt; Nöronlarda ve nasıl davranış testleri kendi işlevini test etmek için. Biz daha fazla farmakolojik test için kullanılabilir birincil nöronal kültürlerin edinme göstermektedir.

Bir<em&gt; Caenorhabditis elegans</emAmylopathy Çalışmaları&gt; Model Sistemi

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

a-caenorhabditis-elegans-model-system-for-amylopathy-study

Research

JoVE Journal

Neuroscience

A Caenorhabditis elegans Model System for Amylopathy Study

10.4K Görüntüleme.  University of Medicine and Dentistry of New Jersey. Biz solucan amylopathies yönlerini incelemek için yöntemler tarif C. elegans. Biz insan Aβ ifade solucanlar oluşturmak için nasıl göstermek 42 Nöronlarda ve nasıl davranış testleri kendi işlevini test etmek için. Biz daha fazla farmakolojik test için kullanılabilir birincil nöronal kültürlerin edinme göstermektedir.

Video: A Caenorhabditis elegans Model System for Amylopathy Study

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Transgenik kullanarak Assaying β-amiloid Toksisite C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Nörobilim

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Antipsikotik İlaçların Etki Mekanizmaları incelenmesi için Yöntemleri<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Bir Model Olarak Koku Sistemi Akson Büyüme Modelleri ve Morfoloji Eğitim için<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Locomotion Speed ​​Etanol Etkilerinin Ölçülmesi için bir Testi<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Uzun süreli Kalsiyum Görüntüleme için Agaroz microchambers<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Roman Metrik Nematodu Embriyonik Uzama karakterize etmek<em&gt; Caenorhabditis elegans</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

bir<emCaenorhabditis elegans</emBeslenme Durumuna Dayalı Bakır Kaçınma Deneyi

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Ratiometric kalsiyum görüntüleme Caenorhabditis Elegans davranışlar içinde bireysel nöronların

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Caenorhabditis elegans'ta Dendritik Dikenleri Görüntüleme

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

Caenorhabditis elegans'ta Dopaminerjik Nöron Morfolojik Değişim ve Dejenerasyon Seviyelerinin Ölçülmesi

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...