Bu çalışma, Ulusal Sağlık Enstitüsü, R01 HG002995 tarafından desteklenen &quot;Zebra balığı Genom Baskı çıtası: Ters Genetik Yaklaşım&quot; CBM.

Bölüm A: Sperm Dondurma Çözümleri 1. Ginsberg Balık Ringers (her 3 günde bir, taze olun; Ginsburg, 1963) NOT: EK SİPARİŞ YAĞIŞ ÖNLEMEK İÇİN ÖNEMLİ 450 ml steril ddH2O çözülür: <ul><li> NaCl 3,25 g </li><li> KCl 0.125 g </li><li> CaCl2 • 2 H2O 0,175 g Daha sonra ekleyin: </li><li> NaHCO3 0.10 g </li></ul> 4 steril ddH2O ve mağaza ° C ile son hacim 500 ml getirin 2. Donma Orta (taze günlük yapın) <ol style="list-style-type:lower-roman"><li> Metanol OLMADAN <ul><li> Ginsburg Balık 10 ml Ringers (oda sıcaklığında). </li><li> Toz Yağsız Süt 1.5 gram </li></ul></li><li> Metanol ile NOT: EK SİPARİŞ YAĞIŞ ÖNLEMEK İÇİN ÖNEMLİ <ul><li> Ginsburg Balık 9 ml (oda sıcaklığında) Ringers </li><li> Metanol 1 ml </li><li> Toz Yağsız Süt 1,5 g </li></ul> Dondurma medya montaj sonra, 20 dakika süreyle iyice karıştırın. orbital çalkalayıcı ya da rocker. Kullanmadan önce, içine kısım 1 ml Eppendorf tüpleri, yüzeyinde kabarcıklar kaçınarak. </li></ol> Bölüm B: sperm dondurmak için gerekli malzemeler Sperm dondurma için gerekli malzemeler <ol><li> 10 ul tek kullanımlık pipetler (Fisherbrand kedi # 22-358.697) </li><li> MESAB / Tricaine içeren balık su için 250 ml bardak </li><li> izlemek camlar (Pyrex kedi # 9985-75) </li><li> P20 Pipetman (veya eşdeğeri) ve ipuçları </li><li> Sünger balık tutucu (sıkma sırasında erkek tutun) </li><li> Yukarıdaki sahne aydınlatma ile mikroskop Diseksiyon </li><li> Düz forseps (örneğin Millipore tipi) </li><li> 2.0ml kriyojenik şişeleri (Corning kedi # 430.488) </li><li> 10X10 cryoboxes (Nalgene kedi # 03-337-7AA) </li><li> Strafor konteyner ile dolu ince ezilmiş kuru ice1 ~ 15 cm </li><li> 15 ml konik tüpler (Falcon # 352.099) </li><li> Sıvı azot içeren Dewar şişesi </li><li> Uzun maşa (mesela, CMS Fisher Sağlık Bakım kedi # 10-316B) </li><li> Cryogloves </li><li> Erkekler için Kirli su tankı </li><li> Bir Eppendorf tüp metanol olmadan dondurma medya ile dolu </li><li> Bir Eppendorf tüp metanol ile dondurma medya ile dolu </li><li> Cryofreezer (örneğin Taylor-Warton K-serisi) </li></ol> 1 ince ezilmiş kuru buz yapmak için basit bir yöntem, bir havlu sarın ve bir çekiç ile ezebileceği . Daha etkili bir yöntem, bir kuru buz değirmeni, örneğin Clawson model RE-2 kullanmaktır. Bölüm C: Sperm Dondurma Yöntemi <ol><li> Kapiller BORULAR MARK: başlangıcına önce, (yani 16.7 mm alttan bkz. Şekil 1.1.) 3,33 ul düzeyinde bir laboratuar kalem ile 10μl kılcal tüpler işareti. </li><li> ERKEK uyutmak MESAB / tricaine (reçete) &quot;Zebra balığı Kitap&quot; balık su içinde seyreltilmesi içeren behere koyun erkek (ler): </li><li> KURU BALIK: Bir kez anestezi, anestezik bir plastik kaşık kullanarak balık kaldırın ve yavaşça ventral tarafında kuruma özel bir dikkat, bir kağıt havlu üzerine kuru balık blot . Lağım etrafında iyice kuruması için önemli böylece su sperm harekete geçirir. Sünger tutucu pozisyon balık, ventral yüzü yukarı (Şekil 1.2). Anal yüzgecinin etrafında bölgede hala bir kimwipe hafifçe kuru, nemli ise. Sperm erken sıkmak değil dikkat edin! </li><li> Place kılcal tüpleri ile birlikte lastik ağız pipet adaptörü kılcal tüp kutladı. Adaptörü, bir ucunda ağızlık ve diğer bir kılcal sahibi olan bir lastik hortum. Alternatif olarak, kılcal tüp, uygun çaplı Tygon boru kısa bir parça ile 50μl Hamilton şırınga bağlı olabilir. </li><li> Place kapsamında erkek ve kılcal tüp sonuna dikkatle kullanarak dışında pelvik yüzgeçler yayılmasını ürogenital açılış maruz. </li><li> SPERM TOPLAR: At pürüzsüz forseps (örneğin Millipore) balık tarafı hafifçe okşayarak tarafından sperm, ya da işaret parmağı ve başparmak arasında balık tarafını hafifçe sıkarak. Nazik vakum (Şekil 1.2) kullanılarak atılır kılcal tüp sperm toplayın. Sperm ile sınırdışı edilebilir dışkı kaçının. </li><li> Ul 3.3 İÇİN SPERM HACMİ normale hacmi, sperm ulaştığında ya da kapiller tüp (yani 16.7 mm veya daha fazla) kalem işareti aşarsa, daha sonra 8. adıma geçin . Sperm hacmi hedef hacim ulaşamazsa, daha sonra kalem işaretine Metanol (Şekil 1.3) OLMADAN Freeze Orta kullanarak hacim normalleştirmek. Kabul edilebilir minimum miktar sperm sperm kalitesi değişir. İyi sperm beyaz ve opak. Düşük sperm sulu görünüyor. 1 ul iyi sperm (ya da 1 / 3 hedef) ve 2 ul sperm (ya da 2 / 3 hedef): Genel olarak, asgari olarak kabul. Cilt şimdi 3.3 ul normalize. </li><li> SPERM İÇİN dölveriminin ADD: M İLE Donma Orta Suckturuncu işareti (Şekil 1.4 yaklaşık toplam hacmi 20 ul) etanol. Kabarcıkları tanıtmak için özel bir dikkat, bir saat camı temiz bir alan üzerine sperm ve dölveriminin karışımı boşaltınız. Yukarı ve aşağı pipetleme ~ 4 kez (kabarcıkları tanıtan kaçının) hafifçe karıştırın. </li><li> 10μl SPERM INTO 2 CRYOVIALS HER YERİ: sperm Pipet 10 ul ilgili bilgileri (Şekil 1.5) etiketli iki ayrı cryovials dibine dölveriminin çözüm. Hızlı, kap şişeleri taşıma ve oda sıcaklığında 15 ml Falcon tüpleri bir arada cryovial / tüp dibine bırakın. Cap Falcon tüp ve ezilmiş kuru buz içine cryovial içeren Falcon tüpleri çifti eklemek. </li><li> 20 MIN İÇİN SPERM FREEZE. KURU BUZ ON: Kuru buz, ince toz şeklinde değil, pelet olmalıdır . Tüpler, sadece kapakları (Şekil 1.6) yeterince derin kuru buz takılı olmalıdır. Kuru buz tüp takip etmek amacıyla, Falcon tüpleri her çift 1-20 (kapaklar üzerindeki yazılı) bir numara vermek ve kuru buz gitmek zamanların rekoru. </li><li> SIVI AZOT INTO YERİ CRYOVIALS: 20 dakika süreyle kuru buz dondurulmuş sperm edildikten sonra, sıvı azot içeren Dewar balon aktarabilirsiniz. Sperm sıvı azot dondurucu içine yerleştirmek saatine kadar burada saklanır. örnekleri ısınmasına kalmamak için sıvı nitrojen banyo dondurucu kutulara yerleştirerek, dondurucu kutusu. Dewar balon şişeleri kurtarmak ve cryogloves başa uzun metal maşa kullanın. Alternatif olarak, eğer dondurucu kutusu, plastik bir kap içinde sıvı azot içinde batırılır tutulur cryovials dondurucu kutusunun içine kuru buz doğrudan transfer edilebilir. </li><li> Kriyojenik sıvı azot dondurucuda saklayın cryovials uzun vadeli. Sperm canlılığı korumak için, şişeleri sıvı nitrojen içinde dalmış saklanır önemlidir. Deneyimlerimize göre, buhar fazında depolanan şişeleri zamanla canlılığını kaybeder. Hız önemlidir! Dölveriminin ekleyerek ve kuru buz sperm şişeleri yerleştirerek (yani adımları 6-10) arasında geçen süre en fazla 30 saniye olmalıdır. </li></ol> Bölüm D: kriyoprezervasyon Sperm Kullanımı, Tüp Bebek Yöntemi Biz bu iki kişi ile en iyi işe yaradığını bulabilirsiniz. Bir kişi, diğer kişinin sperm çözülür kadınlardan yumurta sıkar. <ol><li> Bir su banyosunda 33 ° C&#39;ye ayarlayın </li><li> Sıvı azot içeren Çözülme için hazır olana kadar Dewar balon, sıvı azot dondurucu ve transfer cryovial çıkarın. </li><li> 35mm plastik petri içine kadınlarda yumurta sıkın. Mümkünse, 3 debriyajlar yumurta elde etmek ve bir çanak birleştirmek için deneyin. İlk debriyaj 1 dakika içinde üç iyi kavramalar alamıyorsanız, o zaman (iyi bir kavrama yeterli olacaktır) 4. adıma geçin. Iyi kavrama tanımı:&gt; 150 düzgün güzel görünümlü yumurta (yani sarımsı, beyaz döküntüler ile bozulması göstergesidir). Sperm çözülmüş ise kapalı çanak tutun. </li><li> Sıvı azot flakon çıkarın ve kapağı çıkarın. 33 ° C su banyosunda 8-10 sn hızla batırmayın flakon ~ 1 / 2 yolu. </li><li> Hızla yukarı ve aşağı pipetleme tarafından şişe ve karıştırmak için 70 ul oda sıcaklığında Hanks ekleyin. Hemen yumurta ekleyin ve pipet ucu ile hafifçe karıştırın. </li><li> Gecikmeden 750 ul balık su ekleyerek, sperm ve yumurta etkinleştirin. Çalkalanarak karıştırılır. Oda sıcaklığında 5 dakika inkübe edin. </li><li> 5 dakika sonra, 28 balık, su ve inkübe çanak çanak doldurmak ° C 2-3 saat sonra, sayım ve 100mm yemekleri (50/dish) bereketli embriyolar transfer. Infertil yumurta döllenme frekans tespit edilebilir, böylece güvenin. </li><li> Larvaların iyi dikkat edin! Ilk 5 gün için, günlük su değişimi ve ölü embriyolar kaldırmak. Sadece 5 gün kreş yüzmeye mesanesi larvaları koyun. </li></ol> Bölüm E: Temsilcisi Sonuçlar Biz 2001 ve 2003 yılları arasında toprağın işlenmesinde 8600 dondurulmuş sperm örneklerinin bir kütüphane yapmak için bu crypreservation metodoloji kullanılır. Bu örneklerin 106 çözülmüş ve doğurganlık tespit ettik. Doğurganlık canlı embriyoların in vitro fertilizasyon, dondurulmuş sperm ile döllenmiş yumurta toplam sayısına bölünmesiyle üretilen oranıdır. Taze (cryporeserved olmayan) sperm genellikle&gt;% 95 bereket vardır; Kriyoprezerve sperm, çok daha düşük doğurganlık: bir ortalama% 29 (n = 106 şişeleri). Doğurganlık flakon-flakon değişkenliği,% 1&#39;den daha az% 62 gibi yüksek değişen büyüktür. Bu değişkenlik, standart sapma Şekil temsil eden büyük bir hata çubukları tarafından temsil edilmektedir. 2. Ancak biz sadece bir kez, her iki sperm örnekleri 0% doğurganlık deneyimli ve ilgili toprağın işlenmesinde mutasyon kurtarmak için başarısız bugüne kadar çözülmüş 106 sperm örnekleri. Birkaç yıl boyunca kütüphane sperm şişeleri çözülme (2001-2008) bu yöntemle dondurulmuş sperm örneklerinin ortalama doğurganlık kalmasını buldukistikrarlı bir zaman içinde (Şekil 2). 2001 yılında çözülmüş dondurulmuş sperm örneklerinin ortalama doğurganlık 23.5 (% 11 sperm örnekleri üzerinde) oldu ve 2008 yılında 25 (% 21 sperm örnekleri üzerinde) oldu. Diğer araştırmacılar bir dizi şimdi kendi laboratuarlarında bu sperm dondurma protokolü kullanıyor. Bazı gözlediğimiz benzer doğurganlık oranları ve örnek-örnek değişkenliği ile iyi bir başarı bildirdin. Diğerleri bildirildiğine göre bu protokolü ellerinde çalışmasını sağlamak için başarısız oldu. Biz muhtemel sorunların kaynakları olduğuna inanıyoruz: <ol><li> , Farklı bir kuru buz soğutma oranı yol açan, protokol önerilen tam cryovials veya konik tüpler kullanmak durumunda </li><li> Bir alt-optimal soğutma oranı, toz, kuru buz kullanmak durumunda </li><li> Kötü doğurganlık erkek balık tek bir erkek sperm ul en az 1 almalısınız. Ayrıca taze sperm ile in vitro fertilizasyon yaparak erkek doğurganlığı test edebilirsiniz,% 100 doğurganlık yakın almak için çok az taze sperm alır, çünkü ancak bu yanıltıcı olabilir. </li><li> In vitro fertilizasyon, alt-standart kalitede yumurta kullanılması. Bir şey ama mükemmel debriyajlar kullanılması her zaman,% 0 doğurganlık yakın, aslında iyi doğurganlık (yinelenen flakonun doğurganlık dayalı) olan sperm örnekleri bile yol açar. </li></ol><img src="/files/ftp_upload/1395/1395fig1.jpg" alt="Şekil 1" /> Şekil 1 sperm dondurma yöntemi şematik. <img src="/files/ftp_upload/1395/1395fig2.jpg" alt="Şekil 2" /> Şekil 2 kriyoprezervasyon sperm doğurganlık zaman içinde sabit kalır. Yüzde doğurganlık, dondurulmuş sperm ile döllenmiş yumurta toplam sayısına bölünmesiyle in vitro fertilizasyon üretilen canlı embriyo sayısı. &quot;N&quot;, doğurganlık ilgili yıl içinde test edildi Kriyoprezerve sperm şişeleri sayısını ifade eder. Hata çubukları standart sapma temsil eder. Her sperm flakon farklı bir erkek olduğu için, flakon-flakon değişkenlik görülmektedir.

<ol>
	<li>Ginsburg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm-egg+association+and+its+relationship+to+activation+of+the+egg+in+salmonid+fishes.">Sperm-egg association and its relationship to activation of the egg in salmonid fishes.</a> J. Embryol. Exp. Morphol. 11, 13-33 (1963).</li><li>Harvey, B., Kelley, R. N., Ashwood-Smith, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+zebra+fish+spermatozoa+using+methanol.">Cryopreservation of zebra fish spermatozoa using methanol.</a> Can. J. Zoology. 60, 1867-1870 (1982).</li></ol>

Diğer araştırmacılar bir dizi şimdi kendi laboratuarlarında bu sperm dondurma protokolü kullanıyor. Bazı benzer bereket ve gözlediğimiz aynı örnek örnek değişkenlik bildirdin. Diğerleri bildirildiğine göre bu protokolü ellerinde çalışmasını sağlamak için başarısız oldu. Biz muhtemel sorunların kaynakları olduğuna inanıyoruz: <ol><li> , Farklı bir kuru buz soğutma oranı yol açan, protokol önerilen tam cryovials veya konik tüpler kullanmak durumunda </li><li> Bir alt-optimal soğutma oranı, toz, kuru buz kullanmak durumunda </li><li> Kötü doğurganlık erkek balık tek bir erkek sperm ul en az 1 almalısınız. Ayrıca taze sperm ile in vitro fertilizasyon yaparak erkek doğurganlığı test edebilirsiniz,% 100 doğurganlık yakın almak için çok az taze sperm alır, çünkü ancak bu yanıltıcı olabilir. Daha büyük, daha sağlam renkli erkekler genellikle daha yüksek kalitede sperm verir. </li><li> In vitro fertilizasyon, alt-standart kalitede yumurta kullanılması. Bir şey ama mükemmel debriyajlar kullanılması her zaman,% 0 doğurganlık yakın, aslında iyi doğurganlık (yinelenen flakonun doğurganlık dayalı) olan sperm örnekleri bile yol açar. </li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>10 &mu;l disposable pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-358697</td>
<td>&nbsp;</td>
<tr>
<td>Watch glasses</td>
<td>&nbsp;</td>
<td>Pyrex</td>
<td>9985-75</td>
<td>&nbsp;</td>
<tr>
<td>2 ml cryogenic vials</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430488</td>
<td>&nbsp;</td>
<tr>
<td>10 x 10 cryoboxes</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>03-337-7AA</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>Tongs</td>
<td>&nbsp;</td>
<td>Fisher Health Care</td>
<td>10-316B</td>
<td>&nbsp;</td>
<tr>
<td>Cryofreezer</td>
<td>&nbsp;</td>
<td>Taylor-Warton</td>
<td>K-series</td>
<td>For example</td>
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a high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization

Bu Harvey yöntemi (Harvey ve ark, 1982) bir uyarlaması olan zebrafish sperm dondurulması için bir yöntem. Biz de prosedür düzene ve örnek bütünlüğü artırmak, orijinal protokol iki değişiklik girmiştik. İlk olarak, sperm hacimleri dölveriminin içermez donma medyayı kullanarak tüm normalleştirmek. İkincisi, soğukta saklanan sperm kılcal tüpler yerine cryovials saklanır. (Δ ° C ​​/ saat) sperm dondurma ve çözme oranları muhtemelen bu yordamı kontrol etmek için iki en kritik değişkenler. Bu nedenle, belirtilen bu farklı tüpler yerine geçmez. 2 bu takımlar halinde çalışma ~ 2 saat içinde takım başına 100 erkeklerin sperm dondurmak mümkün. Sperm bu protokolü kullanarak, ortalama% 25 doğurganlık (in vitro fertilizasyon döllenmiş yumurta toplam sayısına bölünmesiyle oluşturulan canlı embriyo sayısı olarak ölçülen) ve bu yüzde doğurganlık, uzun yıllardır istikrarlı dondurularak saklanabilir.

Watch this Scientific Journal Video about A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization at JoVE.com

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

Bu zebrafish yüksek verimlilik için bir sperm dondurulması protokol. Sperm bu protokolü kullanarak,% 25, ​​sonraki vitro fertilizasyon doğurganlık ortalama ve uzun yıllardır istikrarlı dondurularak saklanabilir.

Zebra balığı sperm Kriyoprezervasyon için bir Yüksek Verim Yöntem ve In Vitro Gübreleme

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

a-high-throughput-method-for-zebrafish-sperm-cryopreservation-vitro

Research

JoVE Journal

Biology

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

30.8K Görüntüleme.  University of California, Davis. Bu zebrafish yüksek verimlilik için bir sperm dondurulması protokol. Sperm bu protokolü kullanarak,% 25, ​​sonraki vitro fertilizasyon doğurganlık ortalama ve uzun yıllardır istikrarlı dondurularak saklanabilir.

Video: A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

S. cerevisiae &quot;adlı organel morfolojisi küresel çalışma yüksek verimli bir yöntem

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Biyoloji

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

İnsan pre-mRNA üzerinde Haritalama Ribonucleoproteins Hızlı Yüksek verim için Yöntemi (RNPs)

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice-Cap: Büyüyen Bir Yöntem Arabidopsis</emYüksek Verim Genotiplendirme için 96-iyi Tabaklar ve Domates Tesisleri>

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Yüksek verim Kristalizasyon lipidik Bicelle Yöntemiyle Membran Proteinleri

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

I · Cryo Kit kullanarak Fare Sperm Kriyoprezervasyon ve Kurtarma

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Bilinçli Farelerde Glomerüler Filtrasyon Hızı Ölçümü için bir Yüksek verimli Yöntemi

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

Haploit Zebra balığı Embriyolar üretimi ile<em&gt; In Vitro</em&gt; Gübreleme

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebra balığı Embriyolar kullanılarak Yüksek verim Yaklaşıyor A Manuel Küçük Molekül Ekran

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Yüksek verim Uygulamaları Yetişkin Zebra Hearts Kurtarma

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...