Laboratuarda İş NSF (MCB0919933) ve NIH (GM088790) tarafından desteklenmektedir.

1. Trunk parçası rejenerasyon tahlil <ol><li> Hayvanların atık ve yutulur gıda (~ 30 bozulmamış solucanlar, her solucan ~ açlık sonrası 8-10 mm uzunluğunda) emin olmak için en az 5 gün önce rejeneratif tahlil için solucanlar bir kohort beslenme durdurun. </li><li> Testinin gününde, su ile önceden dondurulmuş tesviye buz bulaşık durulama ve plastik wrap ile düz buzlu bir yüzey kapağı. Forseps kullanarak, plastik ambalaj malzemesi ile bir filtre kağıdı yerleştirin. </li><li> Filtre kağıdı, kaynak suyu birkaç damla ile ıslatın. Planaryalar bir transfer pipet kullanarak filtre (filtre ortalama &lt;20 solucanlar) üzerine aktarın. Worms, gerekirse daha fazla kaynak suyu ile repipetting, filtre yeniden konumlandırılmış olabilir. Aşırı sıvı çıkarın. </li><li> Bir neşter kullanılarak, tek bir kesim hayvan ön apeks ve yutak (Şekil 1A) ön ucu arasında yaklaşık yarım baş organını. </li><li> Bir neşter kullanılarak, yaklaşık yarım kuyruk hayvan sonu ve yutak (Şekil 1A) arka ucu arasında yapılan tek bir kesim tarafından kuyruk bölgesi organını. Neşter kestikten sonra% 70 etanol ile ıslatılmış kağıt havlu ile aşırı mukus çıkarın. Neşter fazla etanol silin. </li><li> Kaynak suyu içeren bir Petri kabı (100 x 25mm derinlik) içine forseps aracılığıyla filtre aktarın. (Yaranın kapanması için, yara pinching ve koyulaşması tarafından gözlemlenebilir) ~ 3 dakika bekleyin. </li><li> Petri kabı içeren termostatlı inkübatör rejeneratif tahlil ve transfer (24 ° C) için istenen orta gövde parçalarının içine filtre kağıdı durulayın. </li><li> ~ 1 hafta sonra yeniden yapılar tanımlanabilir (Şekil 1A) Puan rejeneratif fenotipi. </li></ol> 2. Farmakolojik manipulasyon praziquantel tarafından uyarılan iki kutupluluk: rejenerasyon <ol><li> DMSO çözülebilmesinde ilaç (62.4mg PZQ 200mm hisse senedi için 1 ml) praziquantel (PZQ) stok hazırlayın. Aynı gün kullanın. </li><li> Tamponlu Montjuch tuzları ilaç içeren solüsyon (90 mcM PZQ) 50ml olun. ~ Dağılım sağlamak için 1 dakika karıştırın. </li><li> [Protokol # 1], son adım [1.7] PZQ gövde parçalarının bir kohort açığa, yukarıda ayrıntılı olarak gövde parçası rejeneratif tahlil gerçekleştirin. </li><li> 24-48 saat sonra, döviz PZQ içeren, kaynak suyu için medya solucanlar birkaç kez çamaşır (&gt; 3). </li><li> Inkübatör için solucanlar dönün. Puan iki kutupluluk (iki başkanları, Şekil 1B) bir hafta sonra kesme. </li></ol> 3. Rejenerasyon Genetik manipülasyon: in vivo RNAi besleme protokolü <ol><li> Önce tahlil, tavuk karaciğeri Homojenat hazırlar. Gıda stoklarının gelen yağ, sarı renkli bölümler, ve püresi kalan karaciğer atın. Depolama için örgü süzgeç ve alikotu süspansiyon (1.5mL tüpler, -20 ° C) süzülür püresi. </li><li> Tahlil için, önce 1mL örnekleri -20 ° C&#39;de saklanan, arz aliquoting sığır kırmızı kan hücreleri (eritrositler) hazırlamak </li><li> RNAi için solucanlar seçin. Üç kohortlarında kullanılır: ilgi gen, pozitif kontrol (bilinen RNAi fenotip gen Şekil 1C, D &amp; E) ve negatif kontrol (hiçbir RNAi fenotip gen, aynı zamanda deneysel kohort göre demonte seviyelerinin değerlendirilmesi için yararlı). Her kohort için, ~ 250 solucanlar (en az 5 gün açlıktan sonra uzun ~ 8-10 mm) kullanın. Plastik küvet kaynak suyu saklayın. </li><li> Ilgi dsRNA hedef gen ifade E. coli her RNAi kohort, çözülme stok için [8], tavuk karaciğeri Homojenat bir tüp ile birlikte [3,1] ve eritrositlerde bir tüp [3,2]. </li><li> Pelet bakteriler (13.000 g, 1 dakika). Süpernatant ve tekrar süspansiyon pelet 2xYT medya (~ 700μl) çıkarın. Recentrifuge buz üzerinde supernatant, yer pelet atmak. </li><li> Büyük bir delik P200 pipet ucu sonu keserek oluşturun. RNAi beslenme karışımı oluşturmak için tavuk karaciğeri Homojenat (150 mcL) ve eritrosit (50 mcL) oluşan bir karışım iyice bakteriyel pelet tekrar süspansiyon haline getirin. Santrifüj hava kabarcıklarını çıkarın. </li><li> Besleme için (~ 1 inç derinlikte terk) solucanları içeren plastik küvet suyun çoğunluğu çıkarın. Swirl küvet, solucanlar, bu kabın merkezinde konsantre ve solucanlar corralling bir daire içinde pipet RNAi beslenme karışımı. Diğer Diners rahatsızlık vermesini dikkatlice RNAi beslenme karışımı üzerine geri kaçakla koaksiyel bir transfer pipet kullanın! </li><li> Beslemeden sonra (~ 1 saat), iyi beslenen Planaryalar belirlemek. Bu solucanlar yutulur eritrositlerde koyu kırmızı bir renklenme gösterir. Diğerleri atın. </li><li> Dikkatle, taze bahar su ile çözüm beslenme, gıda egestion önlemek için rahatsızlığı en aza indirmek değiştirin. Bunu yapmak için, hafifçe bir tarafa transfer pipet kullanarak tüp, bulanık su dökmek ve dikkatli bir şekilde küvetin Karşı duvarda dökülen tatlı su ile değiştirin solucanlar yerelleştirmek. Resubmerge Planaryalar hızla havaya uzun süre maruz kalma olarak da gıda egestion sonuçları. </li><li> Gevşek seal konteyner kapağı ve kuvöz dönüş (24 ° C) </li><li> Rejeneratif döngüleri [protokol # 1] RNAi fenotipleri için ekran ile serpiştirilmiş çok sayıda döngüleri üzerinden RNAi besleme protokolü (~ 2-3 gün arayla), tekrarlayın. Şekil 2, toplam süresi yaklaşık 1 ay sürüyor, birçok genin etkili beslenme ve yenilenme için standart bir protokol gösterilmiştir . </li></ol> Optimum protokol mRNA stabilitesini, doku yerelleştirme, protein perdurance, ya da birden fazla besleme döngüleri rejenerasyon sonra önleyen bir fenotip gelişimi gibi faktörlere bağlı olarak, farklı genler için bu şemaya değiştirin. Sadece negatif kontrol kohort ile hedef mRNA düzeylerini karşılaştırmak için fenotipi (görünen eğer) penetrans tarama, veya qPCR yaklaşımlarla tarafından demonte seviyeleri değerlendirin. 4. Temsilcisi sonuçları: Gövde parçası rejenerasyon tahlil tüm solucanlar normal ön-arka (&#39;baş-kuyruk) polarite ile yeniden gerektiği gibi sağlam. Bu kısa sürede, ön eyespots (asterixed Şekil 1A) ortaya çıkması kolaylaştırılmış kesme beş gün sonra, atılır . Ancak, kayıp yapıların morfolojik restorasyon tam bir hafta sonra ortaya çıkar. Iki başlı solucanlar verim PZQ gövde parçası yenilenme Farmakolojik manipulasyon (Şekil 1B) aynı zamanda güçlü (EC 50 = 87 (+ -)% 11 bipolar parçaları, 48 saat 20 70μM PZQ). Iki kutupluluğun tek bir rejeneratif döngüsü boyunca ortaya çıkar. Bu testleri daha düşük etkinlik, büyük olasılıkla uyuşturucu çözünürlük ve / veya depolama, veya PZQ etkisiz bir farklı flatworm türlerin kullanımı ile ilgili sorunlar ile ilgilidir. Penetrasyonun daha düşük olan ilaçlar için, bir anteriorization indeksi genellikle tam iki kutupluluk 22 kenara ara fenotipleri puan için kullanılır. Pozitif kontrol yapıları RNAi fenotipleri Şekil 1&#39;de gösterilmiştir: japonica Dugesia RNAi altı-1 (D j-six-1), gözsüz bir fenotip 23 (Şekil 1C), Dugesia japonica PC2 RNAi (Dj-PC2) sonuçları ışık kaçınma tepkisi 24 (Şekil 1D) ve RNAi kaybı sonuçları Dugesia japonica βcatenin-1 (Dj-βcatenin-1), iki başlı bir gövde parçaları (Şekil 1E) fenotip 16-19, 21 sonuç . Bu fenotipleri aşağıdaki basit RNAi protokolleri kullanarak elde edilebilir: Dj-altı-1 (FFxFx), Dj-PC2 (FFFx), Dj-βcatenin-1 (FFxFx), F = besleme döngüsü ve x = rejeneratif döngüsü sırasıyla. <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="Şekil 1"/> Şekil 1:... Trunk parçası rejenerasyon tahlil Sol, planarian (üstte) ve şematik (orta) Image eksize gövde parçası yerini göstermek için sağ, gövde parçası yenilenme timecourse belirtilen zamanlarda yenileyici parçası görünümünü gösteren (gün) B PZQ maruz tarafından üretilen iki başlı planarian Image Dj-altı-1 RNAi. mobil kontrol solucan göre hareketsiz Dj-PC2 RNAi solucanlar (lekeli kırmızı) ışık kaçınma tepkisi D kaybı, (lekeli tarafından üretilen C &#39;gözsüz&#39; solucan Dj-βcatenin-1 RNAi tarafından üretilen yeşil) E Bipolar planarian. BE, RNAi solucanlar orijinal anterior ucu sola yönelik. <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="Şekil 2"/> Şekil 2: D. birçok genin etkili olduğu demonte için birden fazla beslenmeleri oluşan tipik RNAi protokolü (F) ve rejeneratif döngüleri (x) için devir besleme ve kesim sırası japonica. Bireysel genler için bu protokol Modifikasyonu kullanarak, örneğin, en iyi sonuçlar için gerekli olabilecek daha az (parantez) veya besleme ve rejeneratif döngüleri daha büyük bir sayı.

<ol>
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Çıkar çatışması ilan etti.

Protokolleri japonica planarian Dugesia yenilenme eğitim ve işlenmesi için burada ayrıntılı testlerin nitelendirdi. Bunlar basit ve kolayca laboratuar veya sınıf yapılabilir gibi özel ekipman gerektirmez. Tahliller ayrı ayrı ya da kombine (kimyasal ilaç etkinliklerinin in vivo genetik tarama için) ve aday gen düzeyinde yapılabilir, ya da tarafsız, daha yüksek verim tarama 8 adapte olabilir. Kendi sağ Planaryalar ilginç biyoloji eğitim için olsun, ya da doku rejenerasyonu çalışmak için alternatif bir model memeli homologları in vivo fonksiyonu değerlendirmek için, araştırmacılar bu yaklaşımların çeşitli faiz katalize edilmelidir .

<table border="1"><tbody><tr><th> Reaktif </th><th> Satıcı </th><th> Katalog Numarası </th><th> Yorumlar </th></tr><tr><td> Kaynak suyu </td><td> Kandiyohi. Premium Waters A.Ş. Minneapolis, MN </td><td> n / a </td><td> Kaynak suyu diğer formları da iyi çalışır. Canlılığı testlerin ilk deneme. </td></tr><tr><td> (1,6) NaCl, CaCl2 (1mm), MgSO4 (1mm), MgCl2 (0.1mm), KCl (0.1mm), NaHCO3 (1.2mm), Hepes (1.5mm): 1 x Montjuich tuzları tamponlu. 24 pH 7.4 ° C. </td><td> Birden çok tedarikçiden </td><td> n / a </td><td> PH tampon sağlamak için rejeneratif deneyleri sırasında ilaç tedavileri için yapay su. 5 / 8 Holtfreter çözümü bir alternatiftir. </td></tr><tr><td> 2xYT Broth </td><td> Fisher Scientific </td><td> BP2467-500 </td><td> Medya = 31 g / L. Otoklavlı. </td></tr><tr><td> Petri Dish (100x25mm) </td><td> Fisher Scientific </td><td> 08-757-11 </td><td> Yenilenme döngüleri sırasında Konut solucanlar </td></tr><tr><td> Kare Bulaşık (100x100x15mm) </td><td> Fisher Scientific </td><td> 08-757-11A </td><td> Solucan kesim yüzeyi buz tepsisi olarak kullanılan su, dondurma ile doldurun </td></tr><tr><td> Plastik küvet: Ziploc Büküm &#39;n Loc (16oz). </td><td> Çeşitli perakendeciler </td><td> n / a </td><td> RNAi kohortlarında için uygun su geçirmez kaplarda </td></tr><tr><td> Tavuk Karaciğer </td><td> Ticari bakkal </td><td> n / a </td><td> Antibiyotik kaçınma nedeniyle organik kaynakları önyargılı. </td></tr><tr><td> Blender </td><td> Herhangi bir mutfak tedarikçisi </td><td> n / a </td><td> Tavuk karaciğeri püresi yapmak için kullanın. </td></tr><tr><td> Tel 1mm Mesh süzgeç </td><td> Herhangi bir mutfak tedarikçisi </td><td> n / a </td><td> Tavuk karaciğeri püresi süzme için kullanın </td></tr><tr><td> Sığır kırmızı kan hücreleri </td><td> Lampire Biyolojik Laboratuvarları </td><td> 7240807 </td><td> % 100 eritrosit süspansiyonu Yıkanmış ve havuzlu </td></tr><tr><td> Dairesel filtre kağıtları </td><td> Whatman # 3 </td><td> 1003 055 </td><td></td></tr><tr><td> Transferi Pipet </td><td> Fisher Scientific </td><td> 13-711-41 </td><td></td></tr><tr><td> Steril, cerrahi bıçaklar </td><td> Birden çok tedarikçiden </td><td> n / a </td><td></td></tr><tr><td> ± Praziquantel </td><td> Sigma Aldrich </td><td> P4668 </td><td> 4 karanlıkta saklayın toz alikotları ° C Birbirinden ayrılacak. </td></tr><tr><td> Dj-altı-1 </td><td></td><td> GenBank AJ557022.1 </td><td> RNAi &quot;göz&quot; fenotip 23 için pozitif kontrol </td></tr><tr><td> Dj-βcatenin-1 </td><td></td><td> GenBank AB181913.1 </td><td> Iki başlı fenotip 17 RNAi pozitif kontrol </td></tr><tr><td> Dj-PC2 </td><td></td><td></td><td> RNAi, ışık kaçınma fenotip 24 kaybı için pozitif kontrol </td></tr></tbody></table>

pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica

Serbest yaşayan planarian flatworms olağanüstü rejeneratif kabiliyetleri 1 deneysel kullanım nedeniyle uzun bir geçmişi var. Bu hayvanların eksize Küçük parçaları eksik vücut yapıları yenilenme orijinal vücut planı reformu. Örneğin, eğer sağlam bir solucan bir &#39;gövde&#39; parçası kesilir, yeni bir &#39;kafa&#39; anteriora yeniden ve &#39;kuyruk&#39; (Şekil amputasyon öncesindeki özgün vücut yapılarının &#39;baş-kuyruk&#39; kutupluluk geri posteriora yeniden 1A). Yenileme, &#39;normal vücut homeostazı ve uygulanan doku rejenerasyonu sırasında ~ 30 farklı hücre tipleri haline ayırt neoblasts&#39; olarak bilinen planarian kök hücreleri tarafından tahrik edilmektedir. Bu rejeneratif sürecinin sağlam ve göstermek için kolay. Şimdi birkaç öncü laboratuarları, birçok araç ve fonksiyonel genetik yöntemler bağlılığı sayesinde bu model sistemi için optimize edilmiştir. Sonuç olarak, önemli son gelişmeler planarian gelişimsel plastisite 2-9 temelini oluşturan moleküler olayları anlama ve manipüle yapılmıştır. Planarian model sistem bilim adamlarından oluşan geniş bir ilgi olacaktır. Sinirbilimciler için, model, bütün bir sinir sisteminin rejenerasyon çalışması yerine, sadece tek bir sinir hücresi süreci genellikle birçok kurulan modellerde çalışmanın odak regrowth / tamir için fırsat tanıyor. Planaryalar bir bolluk ifade nörotransmitterlerin (10), merkezi sinir sistemi 11, 12, evrim eğitimi için önemli bir sistem temsil ve davranış tarama potansiyeli 13, 14 . Rejeneratif sonuçlarını, farmakolojik ve genetik apparoaches tarafından manipülasyon için uygun. Örneğin, uyuşturucu, sadece gövde parçaları yerleştirerek amputasyon sonra uyuşturucu içeren farklı zaman noktalarında çözümler rejenerasyon üzerindeki etkileri için taranmalıdır. Mikroenjeksiyon, devir veya dsRNA yapıları 8, 9, 15, bakteriyel ifade beslenme yoluyla da elde edilebilir demonte yöntemleri (in vivo RNAi), bireysel genlerin rolü incelenebilir . Her iki yaklaşım, bipolar hayvanlar 16-21 yüksek penetrans-örneğin, rejenerasyon görsel olarak çarpıcı fenotipleri üretebilir . Bu model ve bu yöntemlerin uygulanması benimsenmesini kolaylaştırmak için, biz, planarian Dugesia japonica kullanarak, farmakolojik ve genetik testlerin (in vivo RNAi ile besleyerek) Bu video makale protokollerde vitrin.

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

, Canlı hayvan içinde kök hücre farklılaşması çalışmak için cazip bir model planarian flatworm. Yenileme, temel laboratuvar kolayca yapılır basit amputasyon deneyleri ile çalışılmış ve farmakolojik ve genetik (uygun<em&gt; In vivo</em&gt; RNAi) Bu makalede protokolleri tarafından ayrıntılı olarak manipülasyon.

Farmakolojik ve Fonksiyonel Genetik Tahliller Planarian Rejenerasyon işleyin<emJaponica> Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

pharmacological-functional-genetic-assays-to-manipulate-regeneration

Research

JoVE Journal

Biology

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

36.7K Görüntüleme.  University of Minnesota Medical School. , Canlı hayvan içinde kök hücre farklılaşması çalışmak için cazip bir model planarian flatworm. Yenileme, temel laboratuvar kolayca yapılır basit amputasyon deneyleri ile çalışılmış ve farmakolojik ve genetik (uygun In vivo RNAi) Bu makalede protokolleri tarafından ayrıntılı olarak manipülasyon.

Video: Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Fareler için açık / koyu Geçiş Testi

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Biyoloji

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

Kortikal nöron: Hücre Tabanlı Tedaviler Laboratuvar gelişmeler geçiş

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Civciv Embriyo Sinir İndüksiyon için Testi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Fareler için Yükseltilmiş Artı Labirent

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Yetişkin Mouse Hipokampal dentat girus diseksiyonu

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Embriyo sırasında Fonksiyonel Artıklık Test Ters Genetik Yaklaşım

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Genetik Dernekleri Fonksiyonel Dayanak Test bir Allel özel Gen İfadesi Testi

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Planarian İmmobilizasyon, Kısmi Işınlama, ve Doku Nakli

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Bakteriyel Fonksiyonel Ağlar ve tanımında Haritalama<em&gt; Escherichia Coli</em&gt; Sentetik Genetik Diziler kullanarak

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

Gen-ifadesi Makinaları Fonksiyonel Tahliller için Küçük ölçekli Nükleer ayıklar

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Farmakolojik ve Fonksiyonel Genetik Tahliller Planarian Rejenerasyon işleyin Dugesia