Yazarlar yılda protokolü geliştirilmiş tüm Jarvis laboratuvar üyelerine teşekkür etmek istiyorum.

<ol>
	<li>Clayton, D. F., Huecas, M. E., Sinclair-Thompson, E. Y., Nastiuk, K. L., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probes+for+rare+mRNAs+reveal+distributed+cell+subsets+in+canary+brain.">Probes for rare mRNAs reveal distributed cell subsets in canary brain.</a> Neuron. 1, 249-261 (1988).</li><li>Wada, K., Sakaguchi, H., Jarvis, E. D., Hagiwara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+glutamate+receptors+in+avian+neural+pathways+for+learned+vocalization.">Differential expression of glutamate receptors in avian neural pathways for learned vocalization.</a> J. Comp. Neurol. 476, 44-64 (2004).</li><li>Haesler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxP2+expression+in+avian+vocal+learners+and+non-learners.">FoxP2 expression in avian vocal learners and non-learners.</a> J. Neurosci. 24, 3164-3175 (2004).</li><li>Jarvis, E. D., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor-driven+gene+expression.">Motor-driven gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 4097-4102 (1997).</li><li>Holzenberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+expression+of+insulin-like+growth+factor+II+in+the+songbird+brain.">Selective expression of insulin-like growth factor II in the songbird brain.</a> J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+analysis+of+chick+embryos+in+whole-mount+and+tissue+sections.">In situ hybridization analysis of chick embryos in whole-mount and tissue sections.</a> Methods Cell Biol. 87, 169-185 (2008).</li><li>Moorman, A. F., Houweling, A. C., de Boer, P. A., Christoffels, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+nonradioactive+detection+of+mRNA+in+tissue+sections:+novel+application+of+the+whole-mount+in+situ+hybridization+protocol.">Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.</a> J. Histochem. Cytochem. 49, 1-8 (2001).</li><li>Horita, H., Wada, K., Rivas, M. V., Hara, E., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dusp1+immediate+early+gene+is+regulated+by+natural+stimuli+predominantly+in+sensory+input+neurons.">The dusp1 immediate early gene is regulated by natural stimuli predominantly in sensory input neurons.</a> J. Comp. Neurol. 518, 2873-2901 (2010).</li><li>Braissant, O., Wahli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+in+situ+hybridization+protocol+using+non-radioactively+labelled+probes+to+detect+abundant+and+rare+mRNAs+on+tissue+sections.">A simplified in situ hybridization protocol using non-radioactively labelled probes to detect abundant and rare mRNAs on tissue sections.</a> Biochemica. 1, 10-16 (1998).</li><li>Kubikova, L., Wada, K., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+receptors+in+a+songbird+brain.">Dopamine receptors in a songbird brain.</a> J. Comp. Neurol. 518, 741-769 (2010).</li><li>Wada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+neuroethological+approach+for+identifying+and+characterizing+a+cascade+of+behaviorally+regulated+genes.">A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 15212-15217 (2006).</li><li>Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+brains+and+a+new+understanding+of+vertebrate+brain+evolution.">Avian brains and a new understanding of vertebrate brain evolution.</a> Nat. Rev. Neurosci. 6, 151-159 (2005).</li><li>Hoke, K. L., Ryan, M. J., Wilczynski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+cues+shift+functional+connectivity+in+the+hypothalamus.">Social cues shift functional connectivity in the hypothalamus.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 10712-10717 (2005).</li><li>Burmeister, S. S., Jarvis, E. D., Fernald, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+behavioral+and+genomic+responses+to+social+opportunity.">Rapid behavioral and genomic responses to social opportunity.</a> PLoS. Biol. 3, e363 (2005).</li><li>Jarvis, E. D., Schwabl, H., Ribeiro, S., Mello, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+gene+regulation+by+territorial+singing+behavior+in+freely+ranging+songbirds.">Brain gene regulation by territorial singing behavior in freely ranging songbirds.</a> Neuroreport. 8, 2073-2077 (1997).</li><li>Jarvis, E. D., Scharff, C., Grossman, M. R., Ramos, J. A., Nottebohm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=For+whom+the+bird+sings:+context-dependent+gene+expression.">For whom the bird sings: context-dependent gene expression.</a> Neuron. 21, 775-788 (1998).</li><li>Feenders, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mapping+of+movement-associated+areas+in+the+avian+brain:+a+motor+theory+for+vocal+learning+origin.">Molecular mapping of movement-associated areas in the avian brain: a motor theory for vocal learning origin.</a> PLoS One. 3, e1768 (2008).</li><li>Chen, C. C., Fernald, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributions+of+two+gonadotropin-releasing+hormone+receptor+types+in+a+cichlid+fish+suggest+functional+specialization.">Distributions of two gonadotropin-releasing hormone receptor types in a cichlid fish suggest functional specialization.</a> J. Comp. Neurol. 495, 314-323 (2006).</li></ol>

Biz ifşa hiçbir şey yok.

MRNA in situ hibridizasyon Radyoaktif yaygın bölgesel doku organizasyonu, hücre tipi ve beyin fonksiyonel aktivite 2-5,10,12-14 eğitim dahil olmak üzere farklı amaçlar için kullanılır. Daha sonra kullanmak olan mRNA beyinde artan sinirsel aktivite, sık sık adı verilen aktivite bağımlı genler veya erken genler bağlıdır genler üzerinde. Yavruların / yenidoğan, gençler de dahil olmak üzere ve çoklu yaş; birden dokularda, beyin, deri ve kas dahil olmak üzere; bu kullanımlar ile, yöntem memeliler (örneğin insan), balık ve amfibiler, kuşlar da dahil olmak üzere, birden fazla tür arasında uygulanmış , yetişkin ve burada bütün embriyo bölümlerde 2,3,5,15-17. Bizim protokolünün özel özellikleri şunlardır: (1) Bu anatomik özgüllük ve nicel özgüllük arasında bir denge oluşturur. X-ray film üzerinde gen ekspresyonu ölçmek için, görüntülerin dijital resimler (. Örn: Şekil 1A ve 1B) almak, Pho kullanınilgi bölgelerinde piksel yoğunluğu ölçmek ve doku dışında film arka plan seviyeleri çıkarmak ama hala cam slayt 2,4 açık olarak toshop (Adobe) histogram fonksiyonu. Hücresel düzeyde ifade ölçmek için, yüksek büyütme (; Şekil 4 40-100X) altında hücreleri üzerinde gümüş taneleri görüntüleri çekin. Daha sonra cam slayt üzerine hücreler olmadan benzer bir alanda arka sayımı dışarı çıkarmak, görüntü gümüş tanelerinin sayısını saymak için NIH at Wayne Rasband tarafından Image J eşik ve tedbir işlevlerini kullanın, hücrelerin sayısına göre bölmek hücre başına ifade. 4,18 bir değerleri (2), aynı anda 100-200 slayt işleme izin, mineral yağ banyosu tarafından oluşturulan sıkı coverslip mühür nedeniyle nispeten yüksek verimlilik olabilir. elde etmek için Standart in-situ hibridizasyon yöntemleri parafilm, oje ve slaytlar çok yer kaplayacak diğer araçlar ile slaytlar imzalamak için uzun sürer; (3) yüksek olduğuDekstran sülfat ve hibridizasyon tamponu 2,13 olarak Denhardt çözümü sayesinde düşük bolluk transkript için hassas; (4) karanlık alan içinde görüntüleme yaklaşımı 4,5 bir mikroskop üzerinde karanlık alan aydınlatma altında fotoğraf çekerken nedeniyle yüksek kontrastlı görüntüler verir. Ayrıca, bu tür faaliyetlere bağlı gen ekspresyonu olarak gen ekspresyonu küçük değişiklikler, hassas algılama 19 özel davranış algı ve üretim sırasında aktive spesifik beyin bölgelerinde belirlemenizi sağlar. Radyoaktif olmayan protokoller göre sınırlamalar ikinci hücresel çözünürlük ve hücre içinde mRNA konumunu daha net olduğunu, ve emülsiyon ile çalışan manipülasyon ve ışığa karşı çok duyarlıdır. Aynı doku 10,17,20 birden fazla genin mRNA etiketlemek için in situ hibridizasyon gibi radyoaktif olmayan gibi diğer yöntemlerle birlikte bizim yöntemi birleştirmek mümkündür. Bu immünsitokimya ile kombine edilebiliramacıyla aynı örnek RNA ve protein ifadesini hem etiketlemek için belirli bir hücre tipleri ile 10 mRNA co-lokalize. Bu çifte etiketleme deneyler için gerekli olan protokol modifikasyonlar bahsedilen referanslarda tarif edilmektedir. Özetle, bizim yaklaşım bölge örgütü, doku fonksiyonel aktivite ve gen fonksiyonu anlamak için zamanlama ve gen ekspresyon hücresel konumun anlamayı kolaylaştırır.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Asetik anhidrit </td><td> VWR </td><td> MK242002 </td><td></td></tr><tr><td> Kloroform </td><td> VWR </td><td> BDH1109 </td><td></td></tr><tr><td> Kresil violet asetat </td><td> Sigma </td><td> C5042 </td><td></td></tr><tr><td> Kriyostat </td><td> Termo bilimsel </td><td> Microm HM550 </td><td></td></tr><tr><td> Deiyonize formamid </td><td> Sigma </td><td> F9037 </td><td></td></tr><tr><td> DTT </td><td> Promega </td><td> P1171 </td><td> 100mM </td></tr><tr><td> EDTA </td><td> Sigma </td><td> ED </td><td></td></tr><tr><td> Kalıp gömülmesi </td><td> VWR </td><td> 15160-215 </td><td></td></tr><tr><td> Fisher marka SuperFrost Plus slayt </td><td> Fisher Scientific </td><td> 22-034-979 </td&gt; <td></td></tr><tr><td> Formaldehit </td><td> VWR </td><td> BDH0506-4LP </td><td></td></tr><tr><td> Formamid </td><td> Sigma </td><td> F7508 </td><td></td></tr><tr><td> GENECLEAN kiti </td><td> Q-Bio gen </td><td> 1001-200 </td><td></td></tr><tr><td> Kodak BioMax MR filmi </td><td> Sigma </td><td> Z350370 </td><td></td></tr><tr><td> Kodak NTB Emülsiyon </td><td> Carestream Health </td><td> 8895666 </td><td></td></tr><tr><td> Kodak Profesyonel geliştirici D19 </td><td> Kodak </td><td> 1462593 </td><td></td></tr><tr><td> Kodak Profesyonel Fixer </td><td> Kodak </td><td> 1971746 </td><td></td></tr><tr><td> β-merkaptoetanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Madeni yağ </td><td> VWR </td><td> IC15169491 </td><td></td></tr><tr><td> NaOH </td><td> VWR </td><td> SX0600-1 </td><td></td></tr><tr><td> Paraformaldehyde </td><td> Sigma </td><td> 76240 </td><td></td></tr><tr><td> Poli A </td><td> Invitrogen </td><td> POLYA.GF </td><td></td></tr><tr><td> RATP </td><td> Promega </td><td> P1132 </td><td> 10mM </td></tr><tr><td> rCTP </td><td> Promega </td><td> P1142 </td><td> 10mM </td></tr><tr><td> rGTP </td><td> Promega </td><td> P1152 </td><td> 10mM </td></tr><tr><td> RNasin </td><td> Promega </td><td> N2111 </td><td> 40Units/μl </td></tr><tr><td> S 35 UTP </td><td> Perkin Elmer </td><td> NEG039C001MC </td><td></td></tr><tr><td> Emniyet-çözün çözüm </td><td> Güvenliği Araştırma Ürünler Uluslararası çözün </td><td> 111177 </td><td></td></tr><tr><td> Sodyum Asetat </td><td> Sigma </td><td> S7899 </td><td> 3M </td></tr><tr><td> Sodyum fosfat dibazik </td><td> Sigma </td><td> S3264 </td><td></td></tr><tr><td> Sodyum fosfat monobazik </td><td> Sigma </td><td> S3139 </td><td></td></tr><tr><td> SP6RNA polimeraz </td><td> Promega </td><td> P1085 </td><td></td></tr><tr><td> Metal raf boyama </td><td> Elektron Mikroskopi Bilimleri </td><td> 70312-54 </td><td></td></tr><tr><td> T7 RNA polimeraz </td><td> Promega </td><td> P2075 </td><td></td></tr><tr><td> Doku-Tek Ekim </td><td> Sakura </td><td> 4583 </td><td></td></tr><tr><td> 5x Transkripsiyon tampon </td><td> Promega </td><td> P1181 </td><td></td></tr><tr><td> Trietanolamin </td><td> VWR </td><td> IC15216391 </td><td></td></tr><tr><td> Tris-HCl (1 M, pH 8,0) </td><td> VWR </td><td> 101449-446 </td><td></td></tr><tr><td> tRNA </td><td> Roche </td><td> 10109509001 </td><td></td></tr></tbody></table> Çözümler: <ol><li> Riboprobes yapımı için Reaktifler: 1.5 ul DNA örneğinin (0.2-0.3 mg / ml), 5x optimize transkripsiyon tampon 2 ul, 100 mM DTT 1 ul (Promega gelen Polimeraz ile gelir, mixayrıca, oda sıcaklığında), 0.3 ul RNasin (40 ünite / ul), AGC ribonukleotid karışım çözeltisi, G 35 UTP 3.5 ul, ve 1 ul RNA polimeraz 1.5 ul. Nükleaz içermeyen su ile 10 ul kadar getir. </li><li> AGC ribonükleotid karışım çözeltisi: 10 mM ATP, GTP ve birlikte CTP eşit miktarda karıştırın. </li><li> 40 ul RNaz ve DNaz ücretsiz su, 3M Sodyum asetat ve% 100 EtOH 125 ul 5 ul: ücretsiz S 35 UTP kaldırmak EtOH yağış Sodyum asetat çözeltisi. </li><li> Hibridizasyon tampon maddesi (10 ml stok):% 100 deiyonize formamid, 5M NaCl 600 ul, 1M tris-HCl (pH = 8.0), 5 ml, 0.5 M EDTA 240 ul (pH = 8.0), 100 kat Denhart çözümü 100 ul, 1M DTT, 20mg/ml tRNA, 20 mg / ml poli A 125 ul, ve sodyum dekstran sülfat ile 1 g, 250 ul 100 ul. 10 ml kadar toplam hacme getirmek için DEPC-arıtılmış su ilave edilir. Tüm sodyum dekstran sülfat eriyene kadar 55 ° C&#39;de inkübe sonra şiddetle çalkalanır ve. Içinde hibridizasyon tamponu Mağaza~ 6 ay boyunca iyi -20 ° C,. </li><li> 4% paraformaldehit çözeltisi tamponlu: paraformaldehit için tasarlanmış, 50 ° C ısıya kadar bir sıcak plaka üzerinde karıştırılarak bir şişeye 760 ml saf suda paraformaldehit ile 40 g ekleyin. Paraformaldehit eritmek için 10N NaOH 320 ul ekleyin. (~ 10 dk) çözüldükten sonra, 10x PBS 100 ml ekleyin ve 1 litre kadar saf su ile ses getirmek. Karıştırın ve paraformaldehit eriyene kadar çözüm ısıtın. PH 7.4 olmalıdır. </li><li> Asetilasyon tamponu: trietanolamin 13.6 ml damıtılmış su artı 1 litre asetik anhidrit 2.52 ml. </li><li> 20x SSPE çözüm: 3M NaCl 200 mM NaH 2 PO 4-H 2 O, ve distile su ile 200 mM EDTA. 10N NaOH ile pH 7.4 &#39;e ayarlamak çözeltisi. </li><li> 10x PBS tamponu: 80g NaCl, KCl 2.0g, Na 2 HPO 4 14.4g, ve KH 2 2.4 g distile su PO 4. PH 7.0 &#39;çözüm ayarlayın ve 1 lite toplam hacmi getirecekr. </li><li> İkinci yıkama solüsyonu:% 0.1 β-merkaptoetanol ve 2x SSPE% 50 formamid </li><li> Cresyl viyole asetat çözeltisi: musluk suyunda% 3 cresyl viyole asetat (damıtılmış su iyi boyama önler), oda sıcaklığında, bir döküm kalıbı içinde karıştırma ile bir gece boyunca eritildi. Bir 1mm Whatman filtre kağıdı ve Büchner hunisi ile suction filtre. </li></ol>

radioactive in situ hybridization for detecting diverse gene expression patterns in tissue

Bir gen ile ifade edilen bir zamanlama, seviye, hücresel yerleşimi ve hücre tipi bilmek genin işlevinin anlayışımıza katkıda bulunmakta. Bu özelliklerin her biri hücre içinde mRNA in situ hibridizasyon ile gerçekleştirilebilir. Burada Clayton ve ark. (1988) 1 özellikle yetişkin omurgalı beyinleri 2-5 için, yıllardır bizim laboratuarımızda başarıyla çalışmaktadır modifiye in situ hibridizasyon yöntemi radyoaktif bir sunuyoruz. Hedef diziyle uzun tamamlayıcı RNA (cRNA) probları düşük bolluk transkript 6,7 tespiti için izin verir. CRNA problar radyoaktif nükleotidlerin Kuruluş düşük bolluğu transkript ve kantitatif analizleri daha da algılama hassasiyetini sağlar, ya da ışığa duyarlı x-ray film veya doku üzerinde kaplı emülsiyon tarafından. Bu yöntemler hedef gen ekspresyonunun uzun vadeli bir kaydın sağlar. Radyoaktif olmayan prob met ile karşılaştırıldığındaBöyle DIG-etiketleme gibi ODS, radyoaktif prob hibridizasyon metodu düşük bolluğu transkriptlerin algılamak için HRP-antikorları ve / veya TSA kiti kullanılarak çok sayıda amplifikasyon adımları gerektirmez. Bu nedenle bu yöntem kantitatif analiz için sinyal şiddeti ve hedeflenen mRNA miktarları arasında doğrusal bir ilişki sağlar. Aynı anda 100-200 slaytlar işleme izin verir. Bu embriyolar farklı gelişim evreleri için de çalışıyor. Embriyonik doku zor doku bölümleri ile hücre popülasyonu arasındaki sınırları görmek için yapım, hücreler arasında daha az uyum ile, yetişkin dokuya göre daha kırılgan olduğu için gen ekspresyonu pek çoğu gelişimsel çalışmaları kısmen insan embriyosu ve radyoaktif olmayan yaklaşımlar 8,9 kullanın. Buna karşılık, hassasiyet büyük aralığı nedeni ile radyoaktif yaklaşımı, daha kolay popülasyonlar arasındaki sınırları görmek için yapım, doku bölgeleri arasında gen ekspresyonu çözünürlükte yüksek kontrast elde edebilmektedir. Bu yöntemi kullanarak, araştırmacılar ortaya koyabilirBir yeni tanımlanan gen mümkün önemi, ve daha da ilgi genindeki fonksiyonu tahmin.

Watch this Scientific Journal Video about Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue at JoVE.com

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Bu protokol başarıyla kantitatif omurgalı türlerin birden çok doku tipleri düzeyleri ve mRNA mekansal kalıpları tespit etmek için kullanılır. Yöntem, düşük bolluk transkript algılamak ve aynı anda slayt yüzlerce işleme izin edebilirsiniz. Biz örnek olarak kuş embriyonik beyin oluşumu ekspresyon profili kullanarak bu protokolü sunarız.

Radyoaktif In situ</emDoku Çeşitli Gen Patterns Saptayan> Hibridizasyon

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

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Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

15.6K Görüntüleme.  Duke University. Bu protokol başarıyla kantitatif omurgalı türlerin birden çok doku tipleri düzeyleri ve mRNA mekansal kalıpları tespit etmek için kullanılır. Yöntem, düşük bolluk transkript algılamak ve aynı anda slayt yüzlerce işleme izin edebilirsiniz. Biz örnek olarak kuş embriyonik beyin oluşumu ekspresyon profili kullanarak bu protokolü sunarız.

Video: Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Biyoloji

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

Radyoaktif olmayan In situ Hibridizasyon Norveç Ladin Uygulanacak Protokol ve Bitki Türlerinin Aralığı

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Active T. in vitro Sulandırma castaneum Telomeraz

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

Yüksek Verimli In situ</emFetal Fare Alt Ürogenital Yolu mRNA İfade Desenler Karakterize> Hibridizasyon Yöntemi

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNA Floresan tarafından Tek hücreli Gen Transkripsiyon Analizi<em&gt; In Situ</em&gt; Hibridizasyon (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Yüksek Çözünürlüklü Tüm Dağı<em&gt; Yerinde</emGen İfade ve Fonksiyon Eğitim için Zebra balığı Embriyolar içinde&gt; Hibridizasyon

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

MicroRNA<em&gt; In situ</em&gt; Formalin Sabit Böbrek Dokular Hibridizasyon

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Radyoaktif In situ Hibridizasyon