Bu eser Ulusal Bilim Fonu tarafından desteklenen ayırt edici genç akademisyenler (81625002), Ulusal Doğa Bilimleri Vakfı Çin (81470389, 81270282, 81601238), Program Shanghai akademik araştırma, lider (18XD1402400), Şangay belediye için Eğitim Komisyonu Gaofeng klinik tıp hibe desteği (20152209), Shanghai Shenkang hastane Geliştirme Merkezi (16CR3034A), Shanghai Jiao Tong Üniversitesi (YG2013MS42), Shanghai Jiao Tong Üniversitesi Tıp (15ZH1003 ve 14XJ10019), Shanghai yelken programı (18YF1413000) ve Bengbu Tıp Fakültesi (Byycx1722) yüksek lisans yenilik programı. Dr. Erhe Gao bizim laboratuarımızda onun önceki yardım için teşekkür ediyoruz.

Tüm hayvan deneyleri Ulusal Sağlık kurallarına Enstitüleri kullanım laboratuvar hayvanları üzerinde göre yapılmıştır ve Enstitü nün hayvan Etik Komitesi tarafından kabul edildi. Erkek C57BL/6J fareler (8-10 hafta yaşındaki) tüm deneyler için kullanılmıştır. Fareler 24 ° C ± 4 ° C, bir 12-h ışık/karanlık döngü, su ve gıda ücretsiz erişim altında patojen-Alerjik koşullarda muhafaza.
1. Adenovirus çözüm hazırlanması
<ol><li>Arıtılmış adenovirus çözüm gelişinden sonra çözüm-80 ° C dondurucuda saklayın. Not: Adenovirus enjeksiyon uygulanabilir farelerde gerçekleştirildi. Adenovirus vektör kısırlık emin olmak için adenovirus (kodlama luciferase, VDR veya VDR hedefleme shRNA) hazırlanmıştır ve ticari olarak9,10saf.</li>
	<li>İşlem gününde,-80 ° C dondurucudan arıtılmış adenovirus çözüm (3 x 1010 plak oluşturan birimler [pfu] /mL) almak ve adenovirus vektör çözüm buz çözme.</li>
	<li>Bir sağlamlılık, steril eldiven ve steril elbise giy.</li>
	<li>50 µL hazırlamak gün operasyon öncesi sterilize Hamilton şırınga. Not: sterilizasyon için Hamilton şırınga gazlı bez içinde sarılmış ve bir yüksek sıcaklık/yüksek basınç sterilizasyon yerleştirilir. "Sterilizasyon masif" modu seçildi ve sterilizasyon "Başlat" düğmesine basarak başladı.</li>
	<li>Tam saf adenovirus çözümü çözdürme sonra % 75 etanol ile adenovirus solüsyon içeren tüp sprey ve adenovirus çözüm laminar akış steril kukuIeta taşıyın.</li>
	<li>Laminar akış steril başlık, Hamilton şırınga 30 G iğne eki tarafından takip bir iğne olmadan Hamilton şırınga kullanarak 50 µL adenovirus çözüm Aspire edin.</li>
	<li>Şırınga 30 G iğne ile yukarı doğru tutun ve iğne (iğne ucu ilk çözüm Damlalar görünümünü tarafından teyit) adenovirus Çözümle doldurulana kadar şırınga pistonu yavaşça itin. Not: Bu 30 G iğne doldurmak için yaklaşık 45 µL çözüm alır hemen hemen hiçbir çözüm şırıngada görünür şimdi.</li>
	<li>Dikkatli bir şekilde başka bir 30 µL adenovirus çözüm Aspire için yukarıdaki hazır şırınga kullanın ve sonraki kullanım için buz üzerinde gevşek kepli şırınga yerleştirin.</li>
</ol>2. anestezi ve ameliyat hazırlığı
<ol><li>20 mL isoflurane isoflurane Buharlaştırıcı ekleyin ve bir oksijen tankı isoflurane Buharlaştırıcı bağlanın.</li>
	<li>Vana açacağız ve oksijen isoflurane Buharlaştırıcı oksijen tankı akış. Oksijen akış hızı 2 L/dk bir oksijen akışını monitörde isoflurane Buharlaştırıcı üzerinden korumak.</li>
	<li>Fare ile % 4 isoflurane % 100 oksijen (2 L/dak) anestezi isoflurane Buharlaştırıcı bağlı bir plastik kafes 2 min için ikna etmek.</li>
	<li>Plastik kafes imzalat fareden çıkar ve yüzükoyun bir laminar akış steril başlıklı plastik bir platform üzerinde güvenli ve anestezi % 2 isoflurane % 100 oksijen (2 L/dak) ile bir burun konisi korumak.</li>
	<li>Yeterli anestezi ayak çimdik yanıt yokluğunda tarafından onaylayın.</li>
	<li>Steril oftalmik krem kurumasını kornealar korumak için her göz için geçerlidir.</li>
	<li>Göğüs ve üst karın tıraş. 1 dk. kaldırmak için depilatory krem piyasada bulunan depilatory krem traş siteye uygulamak ve kürk ile ıslak gauzes kalan.</li>
	<li>Cerrahi sitesi (sol alt kısmında göğüs) iyot-klorheksidin dayalı antiseptik (örneğin entoiodine) 3 scrubs ile sterilize. Steril örtü ile cerrahi sitesi kapak.</li>
</ol>3. Intramyocardial enjeksiyon Adenovirus fare kalbinde
<ol><li>Eldivenleri çıkarma ve steril eldiven yenisini koymak.</li>
	<li>Sterilize forseps, bir mikro-sivrisinek hemostat, Cerrahi makas ve bir gün yüksek bir operasyonda önce iğne bağı sıcaklık/yüksek basınç Sterilizatör (Adım 1.4 nota bakın).</li>
	<li>0,5 cm cilt kesi xiphoid ve koltuk altı bağlanma hattı boyunca olun. Açık açık göğüs büyük ve küçük göğüs kasları forseps ve bir mikro-sivrisinek hemostat ile incelemek.</li>
	<li>İnterkostal alanlarda ortaya çıkarmak büyük göğüs ve göğüs küçük kasları geri çeker. Aracılığıyla işlemek ve dördüncü interkostal boşluk (veya geniş interkostal boşluk gözlem ile) bir mikro-sivrisinek hemostat ile açın.</li>
	<li>Kalp kalp sigara dominant el parmağı ile hafifçe göğüs duvarında sağ tarafına basarak yansıtmak için belgili tanımlık kesme doğru itin.</li>
	<li>Yavaşça externalized kalp sigara dominant el başparmak ve işaret parmağı ile güvenli.</li>
	<li>Toplam 30 µL adenovirus çözeltinin dominant el ile üç siteleri (ventral, dorsal ve lateral duvarına sol ventrikül) adenovirus (şekil 1 c) ile dolu Hamilton şırınga üzerinden sol ventrikül Miyokardiyum içine enjekte et.</li>
	<li>Enjeksiyon tamamlanmasından sonra hemen geri İntratorasik uzaya kalp yerleştirin.</li>
	<li>El ile göğüs duvarı cilt kesi site doğru yavaşça basarak İntratorasik uzayda hava tahliye edin. Not: Başarılı hava tahliye büyük göğüs ve göğüs küçük kasları tarafından sınırdışı hava neden çırparak tarafından kanıtlanan.</li>
	<li>Deri yatay Dikiş dikiş 5-0 ipek sütür ile yakın. Not: Büyük göğüs ve göğüs küçük kasları, onlar sadece açık açık disseke ve onların anatomik yapıları işlemi arada olmadığından sütüre değil.</li>
</ol>4. ameliyat sonrası yönetim
<ol><li>Buprenorfin (3 mg/kg) iki kez günlük işlem11' den sonra ilk 48 saat için operatif ağrı azaltmak için subkutan enjeksiyon yoluyla yönetmek.</li>
	<li>İşlemden sonra hemen tamamen iyileşti kadar fareler bir ısı yastık (37 ° C) üzerinde korumak. Tamamen kurtarır sonra fareyi kafese geri getirin.</li>
	<li>Sterilize kullanılan Hamilton şırınga ve bir yüksek sıcaklık/yüksek basınç sterilizasyon içinde 30 G şırınga iğne (Adım 1.4 nota bakın). Sterilize 30 G şırınga iğne "Sharps" kap içinde toplamak.</li>
</ol>5. kardiyak Luciferase ifade ölçmek için in Vivo görüntüleme
<ol><li>Gün 5 adenovirus enjeksiyon sonra biyoluminesans 15 mg/mL Dulbecco'nın fosfat tamponlu tuz çözeltisi (DPBS), taze stok çözeltisi hazırlamak. Çözüm 0.2 µm filtreden filtre.</li>
	<li>İntra peritoneally uyanık fareler biyoluminesans eriyik vasıl 150 mg/kg vücut ağırlığı ile enjekte.</li>
	<li>Görüntüleme sistemi açın.</li>
	<li>Otomatik görüntüleme sistemi kendi kendini sınama sonra "Işıklı" olarak görüntüleme modunu seçin ve aşağıdaki ayarları yapın: pozlama: otomatik; Binning: 8; FStop: 1; Uyarma: Blok; Emisyon: açık.</li>
	<li>Biyoluminesans enjeksiyon sonra 10 dk, fareler kloral hidrat enjeksiyon (300 mg/kg vücut ağırlığı) anestezi. Yeterli anestezi ayak çimdik yanıt yokluğunda tarafından onaylayın.</li>
	<li>İmzalat fareler bir ısıtmalı görüntüleme sahnede kamera doğru yönetmen göğüs ile yüzükoyun sígara güvenli.</li>
	<li>Görüntüleri (her fare için 1 resim) toplamak ve göğüs aynı dairesel ölçüm bölgeleri (ROI) ilgi çizerek sinyalleri yoğunluğunu ölçmek.</li>
	<li>Fotoğraf topluluğu sonra farelerin kurban ve aşağıdaki bölümünde belirtildiği gibi dokularda toplamak.</li>
</ol>6. hasat dokular
<ol><li>Noktalarda farklı zaman virüs enjeksiyon (şekil 1B) sonra fare ile % 4 isoflurane, anestezi ve anestezi ile % 2 isoflurane burun konisi üzerinden korumak ve yüzükoyun plastik bir platform üzerinde güvenli.</li>
	<li>Ventral Ensizyon ön boyun olun. Şu ortak Karotid arter omohyoid ve boyun kasları geri çeker tarafından maruz.</li>
	<li>Fareler şu ortak Karotid arter transecting ve kan boşaltılmasına kurban.</li>
	<li>Ventral Ensizyon karın içinde olun. Göğüs kafesi midclavicular hattı boyunca her iki kaburga kesme ve diyafram transect.</li>
	<li>Kalp xiphoid kaldırarak maruz. Artan aort bulmak ve Perivasküler yağ dokusu yavaşça çıkarın.</li>
	<li>Aort cannulate ve retrogradely 0.5 mL 4 ° C fosfat tampon çözeltisi (PBS) ile dolu bir 1 mL şırınga bağlı 30 G iğne ile kalp sıvı.</li>
	<li>Perfüzyon sonra kalp tüketim ve sol ve sağ ventrikül bölmek. Karaciğer, akciğer ve dalak tüketim.</li>
	<li>Soğuk PBS (4 ° C) olarak iki yıkamadan sonra toplanan dokulara sıvı azot kriyojenik şişeleri ayrı ayrı saklayın.</li>
</ol>7. Protein ifade tespiti
<ol><li>Homojenizasyon arabellek piyasada bulunan lizis arabellek 1: 100 oranında içine proteaz inhibitörü kokteyl ekleyerek hazırlayın. İşlenecek örnekleri sayısına göre Toplam hacim hesaplamak. Hazırlanan homojenizasyon arabellek sonraki kullanım için 4 ° C'de tutun.</li>
	<li>Sıvı azot tankı dokulardan dışarı çıkar. Yüksek hassasiyetli ölçeklerde (yaklaşık 60 mg doku her parça için) doku tartın.</li>
	<li>Doku için yeni bir 1.5 mL microcentrifuge tüp aktarın. Hemen 200 µL homojenizasyon tampon ve precooled 1.5 mm çelik bilyalar (4 ° C) microcentrifuge tüp içine ekleyin.</li>
	<li>Precooled (4 ° C) microcentrifuge tüplerde otomatik bir doku Bileme Makinası metal sahipleri güvenli ve aşağıdaki ayarlarla makine başlatarak homojenizasyon başlamak: frekans: 70 Hz; süre: 120 s.</li>
	<li>Homojenizasyon sonra homogenate (16.000 × g, 4 ° C) santrifüj kapasitesi ve dikkatle süpernatant yeni 1.5 mL tüp 100 µL pipet ile aktarın.</li>
	<li>Yükleme eklemek tampon çözüm (5 x) süpernatant protein oranı 1:4 ve 5 min için 100 ° C'de protein denatüre.</li>
	<li>Kalp ve diğer organ dokulara protein ifade seviyelerinde daha da Western blot analizi (-den sonuçlanmak göstermek şekil 2C ve şekil 3temsilcisi) tarafından izlenen protokole göre daha önce12açıklanan.</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramyocardial+cell+delivery:+observations+in+murine+hearts.">Intramyocardial cell delivery: observations in murine hearts.</a> J Vis Exp. (83), e851064 (2014).</li></ol>

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Geçerli raporu miyokard infarktüsü indüksiyon bir yönteminden Gao vd tarafından güncellenmiştir kardiyak gen manipülasyon için viral vektörler intramyocardial enjeksiyon için değiştirilmiş bir teknik gösterir 13 şu anda, karakterizasyonu vivo içinde belirli bir gen fonksiyonları en sık dahil nakavt veya transgenik fareler3,14,15,16<s...

Kalp hastalığı morbidite ve mortalite dünya çapında1,2önde gelen nedenidir. Miyokard İnfarktüsü ve kalp yetmezliği gibi ölümcül kalp sorunları için etkili tedavi stratejileri eksikliği temel pathomechanisms yoğun keşfi ve kimliği roman tedavi olanakları3çekiyor. Bu bilimsel keşifler için yaygın olarak kullanılan4,5kalp özel gen manipülasyon olduğunu. Kardiyak gen manipülasyon güçlü transkripsiyon harekete geçirmek gibi efektör nükleaz (TALEN) kullanarak genom düzenleyerek elde edilebilir ve düzenli olarak interspaced kısa palindromik yineler (CRISPR) kümelenmiş / CRISPR ilişkili protein 9 (Cas9) araçları, ya da Ektopik genetik malzemelerin (örneğin, virüs vektörel çizimler proteinler ilgi kodlama genleri taşıyan) teslim edilmesi6. Genom düzenleme kesin ve kronolojik zamanmekansal genom değişiklikler yaşayan fareler sağlar rağmen hala bir zaman alıcı ve emek yoğun pratik6olduğunu. Alternatif olarak,6kalp özel gen manipülasyon virüs vektör veya küçük müdahale RNA (siRNA) karmaşık teslim rutin tarafından gerçekleştirilen.
Virüs vektör teslim yetişkin fare kalp için kabaca iki stratejileri ile elde edilir: sistemik veya yerel enjeksiyon. AAVs AAV9 gibi cardiotropic serotip sistemik enjeksiyon noninvaziv kardiyak belirli bir gen manipülasyon7için. Ancak, bu yöntem vektör verimli iletim ve gen ekspresyonu için gerekli nispeten yüksek miktarda gerektirir ve kas ve karaciğer7gibi nontarget organların önemli iletim neden olabilir. Yerel virüs enjeksiyon intramyocardial enjeksiyon veya intrakoroner teslim7tarafından sağlanır. İntrakoroner teslim virüs intramyocardial enjeksiyon için karşılaştırıldığında kalp içinde daha eşit dağılımı yol açar. Ancak, bu teknik dezavantajları sistemik dolaşım ve iletim nontarget organları8ve onun gereksinimi için basınç ölçüm işlemi sırasında aygıt için viral vektörler dışarı hızlı yıkama vardır. Buna karşılık, viral olarak dağıtabilmenizi daha iyi virüs saklama Miyokardiyum yanı sıra site belirli teslim, ama başarısız intramyocardial enjeksiyon sağlar7vektör. Küçük hayvanlar için intrakoroner teslimat sistemik AAV9 enjeksiyon ve intramyocardial enjeksiyon daha yaygın olarak uygulanan4,5,7iken yapmak teknik olarak zor olur. Sistemik enjeksiyon gerçekleştirmek kolay olsa da, geleneksel intramyocardial enjeksiyon mekanik havalandırma ve Torakotomi gerektiren, geniş doku hasarına neden olur ve zaman alır.
Bu raporda, intramyocardial enjeksiyon için bir kolay, zaman tasarrufu ve yüksek verimli yöntemi açıklanmıştır. Luciferase ve VDR veya VDR, hedefleme shRNA kodlama adenovirus kardiyak gen ekspresyonu işlemek için enjekte ettiler. Hakim sonra gene manipülasyon, aynı zamanda hücreleri ve fare kalp diğer maddeleri enjeksiyonu için bu yöntem kullanılabilir.

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a simple and efficient method for in vivo cardiac-specific gene manipulation by intramyocardial injection in mice

Gene manipülasyon özellikle kalbinde önemli ölçüde soruşturma kalp hastalığı pathomechanisms ve tedavi potansiyellerini artırmak. Vivo kalp özel gen teslim sık sistemik veya yerel teslim tarafından elde edilir. Kuyruk ven yoluyla sistemik enjeksiyon kolay ve verimli kardiyak gen ekspresyonu rekombinant adeno ilişkili virüs 9 (AAV9) kullanarak işleme. Ancak, bu yöntem için verimli iletim vektör nispeten yüksek miktarda gerektirir ve nontarget organ gen iletim neden olabilir. Burada, intramyocardial enjeksiyon vivo içinde kalp özel gen manipülasyon farelerde için bir basit, etkili ve zaman kazandıran yöntemi açıklanmaktadır. Anestezi (havalandırma) olmadan altında göğüs birincil ve ikincil kasları açık açık disseke ve fare kalp hızlı bir şekilde dördüncü interkostal alan küçük bir kesi yoluyla el ile dışa tarafından maruz kalmış. Daha sonra luciferase (Luc) ve vitamin D reseptör (VDR) veya VDR, hedefleme kısa saç tokası RNA (shRNA) kodlama adenovirus Hamilton şırınga Miyokardiyum enjekte ettiler. Sonraki vivo içinde görüntüleme bu luciferase özellikle kalbinden başarılı bir şekilde overexpressed gösterdi. Ayrıca, Western blot analizi başarılı overexpression veya fare kalbinde VDR susturmak doğruladı. Kez hakim, bu teknik gen manipülasyon yanı sıra enjeksiyon hücreleri veya fare kalbinde nanogels gibi diğer malzemeler için kullanılabilir.

Deney Protokolü ve bazı önemli adımlar bildirilen yöntemi için şekil 1' de gösterilmiştir. İntramyocardial enjeksiyon adenovirus kodlama luciferase (Adv-luc) sonra 5 gün, adv-luc enjekte farelerde Imaging vivo içinde kalbinde olan özellikle (şekil 2A, B), luciferase sağlam overexpression belirtti. nontarget organ iletim yokluğu düşündüren Western blot analizi ile (<strong class="xfig"...

Watch this Scientific Journal Video about A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice at JoVE.com

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Burada fareler bir protokol kalp özel gen manipülasyon için mevcut. Anestezi altında fare kalpler dördüncü interkostal uzayda externalized. Daha sonra kodlama belirli genler bir şırınga vivo içinde görüntüleme ve Batı ile protein ifade ölçü tarafından takip Miyokardiyum içine enjekte edildi adenovirüse bakın analiz leke.

Vivo içinde kalp özel gen manipülasyon farelerde Intramyocardial enjeksiyonla için basit ve etkili bir yöntem

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

a-simple-efficient-method-for-vivo-cardiac-specific-gene-manipulation

Research

JoVE Journal

Biology

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

15.7K Görüntüleme.  The First Affiliated Hospital of Bengbu Medical College. Burada fareler bir protokol kalp özel gen manipülasyon için mevcut. Anestezi altında fare kalpler dördüncü interkostal uzayda externalized. Daha sonra kodlama belirli genler bir şırınga vivo içinde görüntüleme ve Batı ile protein ifade ölçü tarafından takip Miyokardiyum içine enjekte edildi adenovirüse bakın analiz leke.

Video: A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Doğru Genotiplendirme için Basit Bir Yöntem

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Biyoloji

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribozom Ciltli Polipeptitler izole bir aracı olarak SECM Tutuklanma sırası kullanılarak

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Testis Hücre Cezalı Hazırlanması için basit ve Verimli Tekniği

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

Farelerde Hızlı noninvaziv Lomber İntratekal enjeksiyon yoluyla Vivo siRNA&#39;nın Transfaksiyon ve Omurilik Gen demonte olarak

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Sendai-virüs ile İnsan Somatik Verimli Üretimi İnsan uyarılmış pluripotent kök hücrelerin

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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

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

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

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

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

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

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Yenidoğan Farelerde intravenöz enjeksiyonlar

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Nükleik Asitlerin süzülmesi İzolasyonu: basit ve hızlı DNA izolasyonu,

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.