Biz tartışmalar ve yorumlar için Skok laboratuar, özellikle Susannah Hewitt, üyelerine teşekkür etmek istiyorum. Bu çalışma Sağlık hibe Ulusal Enstitüsü R01GM086852, RC1CA145746 (JAS) tarafından desteklenmektedir. JAS Bir Lösemi ve Lenfoma Derneği bilgindir. JC Kanser Araştırma Enstitüsü Irvington Enstitüsü üyesidir. MM Ulusal Bilim Vakfı Hibe Bütünleştirici Lisansüstü Eğitim ve Araştırma Staj (NSF IGERT 0333389) tarafından desteklenmektedir.

1. Fluorophores DNA Prob Etiketleme: Nick Çeviri (~ 6 saat) <ol><li> Temizlik BAC DNA (Maxi-prep göre hazırlanmış) ve plazmit veya PCR ürünleri, H2O içinde tekrar süspanse edilir, tüm etiketleme için de kullanılabilir. Not sağlam bir FISH sinyal için, sondalar, en az 10 kb kapsar gerektiğini. </li><li> RNaz DNA inkübe bir 37 30 dakika ° C (Bütün çözeltiler Tablo 1&#39;de listelenmiştir). </li><li> 16. az 2 saat süreyle nick translation reaksiyon inkübe ° C (Tablo 2 ve 3). Doğrudan etiketleme Alternatif yöntemler (: BALIK Etiket, Invitrogen Örnek) kullanılabilir. </li><li> -80 Az bir saat süreyle reaksiyon inaktive -20 ° C&#39;de ° C ya da gece boyunca </li><li> % 2&#39;lik agaroz jel üzerinde prob boyutu belirleyin. Smear yaklaşık 300-500 bp (Cy5-probların smear görünmez olduğunu unutmayın) de çoğunluğu ile, 100 ve 1000 bp arasında olmalıdır. DNA yeteri kadar sindirilmeyen, daha fazla DNA Pol I veDNaz I reaksiyona eklenmiş, ve 16 ° C de iki saat daha inkübe edilebilir </li><li> Karanlıkta iki saat için H 2 0 dolu iki litrelik bardak 0.025 mm filtrelerde probları arındırın. </li><li> Aşağıdaki gibi probları ile bloke etme faktörleri ekleyin: Manşon-1 DNA&#39;sının 10 ug, Hybloc DNA ve 10 ug DNA probları 10 ug için somon sperm 100 ug. -20 ° C&#39;de saklayın DNA probları </li></ol> 2. Prob Yağış / Denatürasyon / Ön tavlama (3-4 saat) <ol><li> 0.3 ug ve DNA probu 1 mg arasında lamel başına kullanılır. 3M NAAC (pH 5.2) ve% 100 etanol, 2.5 hacim, 0.1 hacim ile karıştırın sondalar, -80 az bir saat süreyle inkübe ° C ya da gece boyunca -20 ° C&#39;de ve 4 13,000 rpm&#39;de aşağı dönüş ° C&#39;de 30 dakika süre ile. DNA probu miktarı DNA kalitesi ve hibridizasyon nükleer bölgesine bağlı olarak ayarlanabilir dikkat ediniz. Doğrudan etiketleme Alternatif yöntemler azaltılmış miktarda kullanımına izin verebilirprob. Bir slayt üzerinde birden fazla DNA FISH sinyal tespiti için, farklı prob (tekrar dizileri gibi, ön-tavlanmış olmamalıdır problar hariç) birlikte çökeltilebilir. Aynı zamanda yapılmış olan deneyler için sondalar da bir arada çökeltilebilir. Granüller hafifçe (problar küçük miktarlarda çöktürülür ise, topak görünür olmayan) ikinci floroforlar göre renkli olmalıdır. </li><li> Kuru granül ve 37-45 karanlıkta (Eppendorf bir termomikser kullanılarak) sallama ile lamel başı (bkz. Tablo 4) 15 ul hibridizasyon tamponu içinde tekrar süspansiyon onları ° C (yaklaşık 20 dakika için). </li><li> 75-95 az 5 dakika boyunca denatüre probları ° C. </li><li> 30-60 dakika boyunca 37 ° C de problar Ön tavlama. Ön tavlama uzunluğu probların karmaşıklığı ve kalitesine bağlı olarak ayarlanabilir. </li><li> Gecede hibridizasyon için lamelleri (bakınız Bölüm 3) için sondalar uygulayın. </li></ol> 3. 3Boyutlu DNA FISH - İlk Gün (~ 3 saat) <ol><li> Sigara yapışık hücrelere 1x PBS (lamel başına 5-10 ul 1x PBS ~ 2x10 5 hücre) küçük bir hacimde yoğunlaşmış ve bir poli-L-lisin kaplı lamel ortasına düştü. Bağlanan hücreler, en az 24 saat (~% 70 kesişme noktası) (lamelleri% 0.1 jelatin ile kaplanmış olabilir) lamelleri üzerinde kültive edilmesi gerekmektedir. 18x18 kare için 24-24 mm lamelleri 6 oyuklu plakalar yerleştirilir, ve protokol boyunca, her bir çözelti, iki ml (alternatif olarak yuvarlak 1.5 mm lamelleri 12/24-well plakalar içinde konulabilir) çukur başına kullanılır. </li><li> RT (% 4 PFA stok -20 ° C&#39;de saklanır bölünüp edilebilir) 10 dakika boyunca,% 2 paraformaldehid / 1 x PBS, pH değeri 7-7.4 de hücre düzeltildi. Paraformaldehit (% 16) çözelti halinde satın alınabilir, veya% 4 stok çözeltileri, toz / zerreciklerinin dan hazırlanabilir. Paraformaldehyde toz&gt; 60 ° buz üzerinde C (pH çözülme kolaylaştırmak için 8-9 yükseltilebilir), soğutmalı aşağı ayarlamak az 1x PBS içinde çözülüred pH 7-7.4 için, süzüldü, şahin içinde bölünüp ve -20 ° C de hafta için saklanır </li><li> 1 x PBS içinde üç kez durulama sonra, buz üzerinde 5 dakika boyunca buzlu-soğuk% 0.4 Triton-X-100/1 x PBS içinde hücreler geçirgenliği. Geçirgenliği uzunluğu hücre tipine bağlı olarak ayarlanabilir Not:. DNA FISH immunofloresan ile birlikte ise, immun (bakınız Bölüm 4) bu aşamada yapılmalıdır. </li><li> 37, bir saat boyunca 0.1 mg / 1 x PBS içinde ul RNaz A ° C&#39;de 1x PBS içinde inkübe hücreler üç durulama sonrasında Lameller parafilm ilgili çözüm veya nemli bir odasında bir slayt 100 ul bir damla üzerine hücre yüzü aşağı yerleştirilir. Lameller sonra dikkatlice forseps ile çıkarılır. Herhangi bir direnç karşılaşılırsa, lamelleri hücrelere hasar görmesini önlemek için, 1 x PBS ile su altında olmalıdır. </li><li> 1 x PBS içinde üç kez durulama sonra, buz üzerinde 10 dakika boyunca buzlu-soğuk% 0.7 Triton-X-100 / 0.1 M HCl içinde hücreler geçirgenliği. </li><li> Üç durular sonra in 1 x PBS, oda sıcaklığında 30 dakika için 1.9 M HCl içinde denatüre hücreler. Seçenek olarak ise, hücreler en az 30 dakika süre ile 75-80 ° C sıcaklıkta% 50 formamid / 2 x SSC içinde denatüre edilebilir olabilir. Hibridizasyon uzunluğu (ve sıcaklık) hücre tipine bağlı olarak optimize edilebileceğini not edin. </li><li> Buz 1x PBS içinde üç durular sonra, ° C karanlık ve nemli odasında 37 gecede probları ile hücrelerin melezleşme. Lameller bir slayt üzerinde prob 15 ul damla üzerine hücre yüzü aşağı yerleştirilir ve lastik çimento ile mühürlenir. </li></ol> 4. 3 boyutlu Immuno-BALIK - İlk Gün (~ 6-7 saat) <ol><li> Kombine immünofloresan / DNA balık, immüno-leke% 0.4 Triton-X-100 (Basamak 3.3) içinde geçirgenliği sonra yapılmalıdır. </li><li> Oda sıcaklığında 30 dakika (% 2.5 bovin serum albümini (BSA),% 10 normal keçi serumu,% 0.1 Tween-20) için solüsyon engelleme hücreler inkübe edin. Bloke etme çözeltisi, filtre edilmiş 1.5 ml Eppendorfs içinde bölünüp m depolanır-20 ° C&#39;de onths </li><li> Bir nemli oda içinde oda sıcaklığında bir saat boyunca, protein ya da bloke edici çözelti içinde seyreltilmiş ilgi histon modifikasyonu karşı yükseltilmiş birincil antikor ile hücreler inkübe edin. Burada H2AX (; ayrıntılar için bakınız Tablo 5 γ-H2AX, Millipore) ve fosforlu serin-139 karşı bir antikor örneğini gösterir. Lameller parafilm ilgili çözüm veya nemli bir odasında bir slayt 100 ul bir damla üzerine hücre yüzü aşağı yerleştirilir. Lameller sonra dikkatlice forseps (1 x PBS, gerekirse su basması) ile çıkarılır. Not seyreltme ve kuluçka süresini antikor ve hücre-tipi kalitesine bağlı olarak ayarlanabilir olabilir. </li><li> % 0.2 BSA /% 0.1 Tween-20/1 x PBS, sallama ile oda sıcaklığında üç kez 5 dak. Yıkayın hücrelerini </li><li> Karanlık ve nemli odasına RT az bir saat için bir çözüm engelleme seyreltilmiş uygun fluorofor-konjuge sekonder antikor hücreleri inkübe edin. Burada bir algılama ile bir örneğini gösterirİkinci keçi anti-fare antikoru (Alexa Fluor 488 veya 555, Invitrogen; ayrıntılar için Tablo 5&#39;e bakınız). Hapten-konjuge sekonder antikor (biotin, digoxigenin, vb) de kullanılabilir. Bu durumda, uygun algılama (streptavidin, anti-Dig, vs) için ilave bir adım aşırı gece DNA-FISH hibridizasyon önce ya da sonra gerçekleştirilebilir. </li><li> % 0.1 hücreleri yıkayın Tween-20 / 1x PBS, karanlıkta sallayarak RT az üç kere 5 dk. </li><li> Karanlıkta oda sıcaklığında 10 dakika boyunca% 2 paraformaldehid / 1 x PBS içinde post-fix hücreler. </li><li> Yukarıda RNaz A (Adım 3.4) inkübasyon DNA BALIK devam edin. </li></ol> 5. 3 boyutlu DNA FISH / Immuno-BALIK - İkinci Gün (~ 2 saat) <ol><li> Dikkatlice dikkatle forseps ile çıkarın ve yıkama çözeltisi içeren kuyularda koyun, 2x SSC, sel lamelleri kauçuk çimento çıkarın. </li><li> 37 30 dak ° sallayarak karanlıkta C 2x SSC hücreleri yıkayın.</li><li> Sallama ile karanlıkta oda sıcaklığında 30 dakika için 2x SSC içinde hücreleri yıkayın. </li><li> Sallama ile karanlıkta oda sıcaklığında 30 dakika süre ile 1 x SSC içinde yıkayın hücrelerini formamid denatürasyon durumunda, yıkama aşağıdaki ile değiştirilmesi gerektiğini not:. 3 yıkamaya 2x SSC, 5,% 50 formamid / 2 x SSC ve 3 yıkar 37, her bir dak ° C. </li><li> Toplam DNA counterstaining için 1.5 ug / ml DAPI (4,6-diamidino-2-fenilindol) ihtiva eden Altın montaj orta uzatmak hücreleri monte edin. Lameller bir slayt üzerinde orta montaj 10-15 ul damla üzerine hücre yüzü aşağı yerleştirilir ve oje ile mühürlenir. Slaytlar -20 ° C&#39;de ay tutulur </li></ol> 6. 3D Mikroskopi ve Analiz <ol><li> 3D görüntüler farklı mikroskop sistemleri ile elde edilebilir. Deneylerde, 0,3 mikron ile ayrılmış optik bölümleri bir Leica SP5 AOBS konfokal sistemi (Acousto-Optik Beam Splitter) üzerinde toplanır. Unutmayın optika arasındaki mesafeyil düzlemleri analiz tipine bağlı olarak ayarlanabilir. </li><li> 3D görüntü yığınlarından farklı yazılım ve araçları kullanılarak analiz edilebilir. Biz faiz lokusların arasındaki mesafe ölçümlerini değerlendirmek ve alt nükleer bölmeleri veya proteinler ile ilgi lokusların dernek farklı analiz Image J yazılımı kullanın. </li><li> Farklı hücre tipleri veya aşamaları arasında karşılaştırma, farklı ilgi lokusları arasında karşılaştırılması: Tüm istatistiksel analizler anlamlılık pairwise değerlendirmesi (ler) ile (veya daha fazla) farklı biyolojik koşulların ikisini karşılaştırmak için yapılmaktadır. Tüm istatistiksel testlerde, P değerleri ≤ 5.00E-2 (a = 0.05) (1.00E-2 olarak anlamlı alınır ≤ p ≤ 5.00E-2 * anlamlı; 1.00E-3 ≤ p ≤ 1.00E-2 * * çok önemli; P &lt;1.00E-3 *** çok önemli). </li></ol> Iki allel arasındaki mesafeleri tüm dağılımlarının istatistiksel analizi. Biz ilk cBiz nonparametrik iki örnekli Kolmogorov-Smirnov (KS) testi 13,14 kullanabilirsiniz ampirik inter-allelik mesafelerin dağılımları farklılıklar önemini değerlendirmek iki alleles.To arasında ölçülen uzaklıkları tüm aralığında kümülatif dağılım frekans eğrileri onstruct. Optimal bir mesafe kesme kullanarak iki allel yakın ilişki (yani eşleştirme) istatistiksel analizi. İki allel veya lokus arasındaki mesafelerde en sağlam farklılıkların aralığını belirlemek için, biz iki kuyruklu Fisher testi 15 s bir dizi kullanın. Yani, her ölçülen mesafe bir şartla anlamlı iki örnek arasındaki kısa mesafelerde daha fazla temsil edilip biz testi. P-Değerler dağıtımında en az iki alleli yakın ilişki (yani eşleştirme) için bir cut-off olarak kabul edilmelidir mesafeleri aralığını gösterir. Ardından iki kuyruklu Fisher exact t testi kullanılırtanımlanan kesim değeri 15 iki allel eşleştirme önemini değerlendirmek o. Bu çoklu test düzeltmeler yapılmıştır 16 Fisher test toplam sayısı için hesap uygulanır. Alt-nükleer bölmeleri veya proteinler ile ilgi odağı ilişkisi istatistiksel analizi. İki kuyruk Fisher-exact testi farklı bölmeleri veya proteinler 15 ile ilgi odağı dernek önemini analiz etmek için kullanılır.

<ol>
	<li>Chaumeil, J., Okamoto, I., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-chromosome+inactivation+in+mouse+embryonic+stem+cells:+analysis+of+histone+modifications+and+transcriptional+activity+using+immunofluorescence+and+FISH.">X-chromosome inactivation in mouse embryonic stem cells: analysis of histone modifications and transcriptional activity using immunofluorescence and FISH.</a> Methods in enzymology. , 376-405 (2004).</li><li>Cremer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+3D+fluorescence+in+situ+hybridization+for+imaging+interphase+chromosomes.">Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes.</a> Methods Mol. Biol. 463, 205-239 (2008).</li><li>Chaumeil, J., Augui, S., Chow, J. C., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+immunofluorescence,+RNA+fluorescent+in+situ+hybridization,+and+DNA+fluorescent+in+situ+hybridization+to+study+chromatin+changes,+transcriptional+activity,+nuclear+organization,+and+X-chromosome+inactivation.">Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation.</a> Methods Mol. Biol. 463, 297-308 (2008).</li><li>Solovei, I., Cremer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-FISH+on+cultured+cells+combined+with+immunostaining.">3D-FISH on cultured cells combined with immunostaining.</a> Methods Mol. Biol. 659, 117-126 (2010).</li><li>Markaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+3D-FISH+and+super-resolution+structured+illumination+microscopy+for+studies+of+3D+nuclear+architecture:+3D+structured+illumination+microscopy+of+defined+chromosomal+structures+visualized+by+3D+(immuno)-FISH+opens+new+perspectives+for+studies+of+nuclear+architecture.">The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture.</a> BioEssays : news and reviews in molecular, cellular and developmental biology. 34, 412-426 (2012).</li><li>Heard, E., Bickmore, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+gene+regulation+and+chromosome+territory+organisation.">The ins and outs of gene regulation and chromosome territory organisation.</a> Current opinion in cell biology. 19, 311-316 (2007).</li><li>Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+sequence:+cellular+organization+of+genome+function.">Beyond the sequence: cellular organization of genome function.</a> Cell. 128, 787-800 (1016).</li><li>Fraser, P., Bickmore, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+organization+of+the+genome+and+the+potential+for+gene+regulation.">Nuclear organization of the genome and the potential for gene regulation.</a> Nature. 447, 413-417 (2007).</li><li>Cremer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+territories--a+functional+nuclear+landscape.">Chromosome territories--a functional nuclear landscape.</a> Current opinion in cell biology. 18, 307-316 (2006).</li><li>Mao, Y. S., Zhang, B., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+function+of+nuclear+bodies.">Biogenesis and function of nuclear bodies.</a> Trends in genetics : TIG. 27, 295-306 (2011).</li><li>Dostie, J., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+organization+in+the+nucleus+-+charting+new+territory+across+the+Hi-Cs.">Chromosome organization in the nucleus - charting new territory across the Hi-Cs.</a> Current opinion in genetics &amp; development. 22, 125-131 (2012).</li><li>van Steensel, B., Dekker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics+tools+for+unraveling+chromosome+architecture.">Genomics tools for unraveling chromosome architecture.</a> Nature. 28, 1089-1095 (2010).</li><li>Massey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kolmogorov-Smirnov+Test+for+Goodness+of+Fit.">The Kolmogorov-Smirnov Test for Goodness of Fit.</a> Journal of the American Statistical Association. 253, 1951 (1951).</li><li>Collins, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RUNX+transcription+factor-mediated+association+of+Cd4+and+Cd8+enables+coordinate+gene+regulation.">RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation.</a> Immunity. 34, 303-314 (2011).</li><li>Fisher, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+interpretation+of+&#967;2+from+contingency+tables,+and+the+calculation+of+P.">On the interpretation of &#967;2 from contingency tables, and the calculation of P.</a> Journal of the Royal Statistical Society. 85, 87-94 (1922).</li><li>Benjamini, Y. H., Yosef, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+false+discovery+rate:+a+practical+and+powerful+approach+to+multiple+testing.">Controlling the false discovery rate: a practical and powerful approach to multiple testing.</a> Journal of the Royal Statistical Society, Series B (Methodological). 57, 125-133 (1995).</li><li>Fitzsimmons, S. P., Bernstein, R. M., Max, E. E., Skok, J. A., Shapiro, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+accessibility,+nuclear+positioning,+recombination,+and+transcription+at+the+Igkappa+locus.">Dynamic changes in accessibility, nuclear positioning, recombination, and transcription at the Igkappa locus.</a> J. Immunol. 179, 5264-5273 (2007).</li><li>Fuxa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax5+induces+V-to-DJ+rearrangements+and+locus+contraction+of+the+immunoglobulin+heavy-chain+gene.">Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene.</a> Genes Dev. 18, 411-422 (2004).</li><li>Goldmit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+ontogeny+of+the+Igk+locus+during+B+cell+development.">Epigenetic ontogeny of the Igk locus during B cell development.</a> Nature. 6, 198-203 (2005).</li><li>Hewitt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+the+Igk+and+Igh+immunoglobulin+loci+mediated+by+the+3'+Igk+enhancer+induces+'decontraction'+of+the+Igh+locus+in+pre-B+cells.">Association between the Igk and Igh immunoglobulin loci mediated by the 3' Igk enhancer induces 'decontraction' of the Igh locus in pre-B cells.</a> Nature. 9, 396-404 (2008).</li><li>Johnson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+Functionally+Segregates+the+Pro-B+Cell+Stage+by+Regulating+Transcription+of+Recombination+Mediators+across+Cell+Cycle.">IL-7 Functionally Segregates the Pro-B Cell Stage by Regulating Transcription of Recombination Mediators across Cell Cycle.</a> Journal of Immunology. , (2012).</li><li>Karnowski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+and+nuclear+repositioning+of+the+lambda5+gene+locus+at+the+pre-b+cell+stage+requires+Aiolos+and+OBF-1.">Silencing and nuclear repositioning of the lambda5 gene locus at the pre-b cell stage requires Aiolos and OBF-1.</a> PLoS ONE. 3, e3568 (2008).</li><li>Kosak, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subnuclear+compartmentalization+of+immunoglobulin+loci+during+lymphocyte+development.">Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development.</a> Science. 296, 158-162 (2002).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+is+a+critical+regulator+of+B-cell+development.">Yin Yang 1 is a critical regulator of B-cell development.</a> Genes Dev. 21, 1179-1189 (2007).</li><li>Parker, 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=The+pre-B-cell+receptor+induces+silencing+of+VpreB+and+lambda5+transcription.">The pre-B-cell receptor induces silencing of VpreB and lambda5 transcription.</a> Embo J. 24, 3895-3905 (2005).</li><li>Roldan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locus+'decontraction'+and+centromeric+recruitment+contribute+to+allelic+exclusion+of+the+immunoglobulin+heavy-chain+gene.">Locus 'decontraction' and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene.</a> Nature immunology. 6, 31-41 (2005).</li><li>Skok, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonequivalent+nuclear+location+of+immunoglobulin+alleles+in+B+lymphocytes.">Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes.</a> Nature. 2, 848-854 (2001).</li><li>Skok, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+contraction+by+looping+of+the+Tcra+and+Tcrb+loci+in+rearranging+thymocytes.">Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes.</a> Nature immunology. 8, 378-387 (2007).</li><li>Xiang, Y., Zhou, X., Hewitt, S. L., Skok, J. A., Garrard, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifunctional+element+in+the+mouse+Igkappa+locus+that+specifies+repertoire+and+Ig+loci+subnuclear+location.">A multifunctional element in the mouse Igkappa locus that specifies repertoire and Ig loci subnuclear location.</a> Journal of Immunology. 186, 5356-5366 (2011).</li><li>Hewitt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAG-1+and+ATM+coordinate+monoallelic+recombination+and+nuclear+positioning+of+immunoglobulin+loci.">RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci.</a> Nature immunology. 10, 655-664 (2009).</li><li>Deriano, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RAG2+C+terminus+suppresses+genomic+instability+and+lymphomagenesis.">The RAG2 C terminus suppresses genomic instability and lymphomagenesis.</a> Nature. 471, 119-123 (2011).</li><li>Brown, K. E., Baxter, J., Graf, D., Merkenschlager, M., Fisher, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+repositioning+of+genes+in+the+nucleus+of+lymphocytes+preparing+for+cell+division.">Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division.</a> Molecular cell. 3, 207-217 (1999).</li><li>Fernandez-Capetillo, O., Lee, A., Nussenzweig, M., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+the+histone+guardian+of+the+genome.">H2AX: the histone guardian of the genome.</a> DNA repair. 3, 959-967 (2004).</li><li>Croft, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+localization+and+morphology+of+chromosomes+in+the+human+nucleus.">Differences in the localization and morphology of chromosomes in the human nucleus.</a> The Journal of cell biology. 145, 1119-1131 (1999).</li><li>Chaumeil, J., Le Baccon, P., Wutz, A., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+Xist+RNA+in+the+formation+of+a+repressive+nuclear+compartment+into+which+genes+are+recruited+when+silenced.">A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.</a> Genes Dev. 20, 2223-2237 (2006).</li><li>Walter, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+many+colors+in+FISH+on+3D-preserved+interphase+nuclei.">Towards many colors in FISH on 3D-preserved interphase nuclei.</a> Cytogenetic and genome research. 114, 367-378 (2006).</li><li>Toomre, D., Bewersdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+wave+of+cellular+imaging.">A new wave of cellular imaging.</a> Annual review of cell and developmental biology. 26, 285-314 (2010).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> The Journal of cell biology. 190, 165-175 (2010).</li><li>Dobbie, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMX:+a+new+platform+for+multimodal,+multichannel+wide-field+imaging.">OMX: a new platform for multimodal, multichannel wide-field imaging.</a> Cold Spring Harbor protocols. , 899-909 (2011).</li><li>Boyle, S., Rodesch, M. J., Halvensleben, H. A., Jeddeloh, J. A., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+with+high-complexity+repeat-free+oligonucleotide+probes+generated+by+massively+parallel+synthesis.">Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis.</a> Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 19, 901-909 (2011).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Yukarıda ayrıntılı teknikler lenfositler 30,31 geliştirme İmmunoglobulin ve Tcra / d loci V (D) J rekombinasyon düzenleme analiz etmek için, laboratuar kullanılmıştır. Biz bu tekniği farklı hücre tiplerinde çeşitli nükleer proteinler, nükleer bölmeleri ve lokus tespiti için adapte edilebilir eminiz. Protokol değişiklikler gerekli olabilir, ve bu durumda, odaklanmak için önemli adımlar aşağıdaki gibidir. İlk olarak, geçirgenliği uzunluğu hücre tipine bağlı ...

Epigenetik mekanizmalar tetik kuruluş ve gelişim ve hücre tipi özel transkripsiyonel profilleri kalıtım. Bir düzeyde bu aktif veya sessiz genomik bölgelerde tanımlar kromatin paketlenmesi modülasyonu içerir. Daha büyük ölçekte, genom ve nükleer mimarisinin küresel 3D organizasyonu da transkripsiyonel desen kontrolünde bir rol oynamaktadır. Böylece, bu epigenomic özelliklerin diseksiyonu genler 6-11 düzenlenir nasıl tam bir anlayış için önemlidir. Kombine immünofloresan ve 3D DNA FISH spesifik etkileşimler / çekirdeğin içinde DNA dizileri ve / veya protein dernekler değerlendirerek, biyokimyasal ve moleküler analizler tamamlamak için eşsiz bir fırsat sunmaktadır. Bunun yanında, bu tür kromatin immünopresipitasyon (ChIP-seq) veya derin dizileme ile birleştiğinde kromozom yakalama konformasyon olarak genom yüksek verimlilik teknikleri (4C-seq, 5C, Hi-C) küresel dat sağlamakhücre popülasyonlarının 12 üzerinde, immünofloresan / DNA FISH teknikleri tek hücre düzeyinde analiz sağlar. Burada 3D mikroskopi ve analizler (3D immuno-FISH) izledi DNA FISH immünofloresan ve DNA dizileri tarafından histon modifikasyonları eşzamanlı tespiti için bir protokol açıklar. Bu protokolün avantaj DNA ve protein yapılarının korunması kombine görselleştirme olduğunu. Bu alanda tecrübemiz mevcut protokoller geliştirmek ve kolaylaştırmak için bize sağladı. Bu rekombinasyon geçiren lenfosit çift-sarmallı DNA mola tespit etmek için bu protokol kullanılmıştır, ancak bu yöntem, diğer proteinlere ve diğer hücre türleri uygulanabilir.

<table border="1"><tbody><tr><td> Reaktif / Malzeme Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> H2O </td><td> Balıkçı </td><td> # BP2470 </td><td></td></tr><tr><td> RNaz A </td><td> Sigma </td><td> # R4642 </td><td></td></tr><tr><td> dNTP </td><td> Sigma </td><td> # DNTP100 </td><td></td></tr><tr><td> Alexa dUTP </td><td> Invitrogen </td><td> # C11397 C-11401 </td><td></td></tr><tr><td> Cy3 veya Cy5, dUTP </td><td> Balıkçı </td><td> # 45-001-xxx </td><td></td></tr><tr><td> DNaz I </td><td> Roche </td><td> # 04536282001 </td><td></td></tr><tr><td> DNA Pol I </td><td> Biolabs </td><td> # M0209 </td><td></td></tr><tr><td> 0.025 mikron filtre </td><td> MilliporE </td><td> # VSWP02500 </td><td></td></tr><tr><td> Manşon-1 DNA 1 mg / ml </td><td> Invitrogen </td><td> # 18440 </td><td></td></tr><tr><td> Hybloc DNA 1 mg / ml </td><td> Uygulamalı Genetik </td><td> # MHB </td><td></td></tr><tr><td> Somon sperm </td><td> Sigma </td><td> # D1626 </td><td> toz H2O içinde 10 mg / ml &#39;de tekrar süspanse edilmesi </td></tr><tr><td> NAAC (Sodyum Asetat, pH 5.2, tampon çözeltisi) </td><td> Sigma </td><td> # S7899 </td><td></td></tr><tr><td> Ficoll 400 (Mol Biol grade) </td><td> Balıkçı </td><td> # 525 </td><td></td></tr><tr><td> Polivinilpirolidon (Mol Biol grade) </td><td> Balıkçı </td><td> # BP431 </td><td></td></tr><tr><td> Dekstran sülfat tozu </td><td> Sigma </td><td> # D8906 </td><td></td></tr><tr><td> SSPE (Tuzlu-Sodyum Fosfat-EDTA) 20x çözümü </td&gt; <td> Balıkçı </td><td> # BP1328 </td><td></td></tr><tr><td> Formamid </td><td> Balıkçı </td><td> # BP227 </td><td></td></tr><tr><td> Lameller </td><td> Balıkçı </td><td> # 12-548-B </td><td></td></tr><tr><td> Slaytlar </td><td> Balıkçı </td><td> # 12-550 </td><td></td></tr><tr><td> 6-well plate </td><td> Balıkçı </td><td> # 0720080 </td><td></td></tr><tr><td> 10x PBS, </td><td> Balıkçı </td><td> # MT-46-013-CM </td><td></td></tr><tr><td> Poli-L-lisin çözelti </td><td> Sigma </td><td> # P8920 </td><td></td></tr><tr><td> Paraformaldehyde, zerreciklerinin,% 95 </td><td> Sigma </td><td> # 441244 </td><td></td></tr><tr><td> Triton-X-100, Mol Biol dereceli </td><td> Sigma </td><td> # T8787 </td><td></td></tr><tr><td> BSA (Bovine Serum Albumin) Fraksiyon V </td><td> Balıkçı </td><td> # BP 1600 </td><td></td></tr><tr><td> Normal keçi serumu </td><td> Vektör Labs </td><td> # S-1000 </td><td></td></tr><tr><td> Tween-20, Mol Biol dereceli </td><td> Sigma </td><td> # P9416 </td><td></td></tr><tr><td> SSC (Salin Sodyum Sitrat) 20x çözümü </td><td> Balıkçı </td><td> # BP1325 </td><td></td></tr><tr><td> Altın antifade reaktifi uzatmak </td><td> Invitrogen </td><td> # P36930 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-diamidino-2-fenilindol) </td><td> Sigma </td><td> # D9542 </td><td></td></tr><tr><td> En testi tek kat kauçuk çimento </td><td> Sanat veya ofis malzemeleri mağazaları </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tablo 1. Özgül reaktifleri ve küçük ekipman. </td></tr></tbody></table>

combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization

3D konfokal mikroskopi ardından 3 boyutlu korunmuş çekirdekleri üzerinde DNA problar kullanılarak floresan in situ hibridizasyon (3D DNA FISH) bireysel hücrelerin alt bölgeler kromozomal gen lokuslarının yeri veya tüm ülkelerle görselleştirmek için en doğru yolu gösterir. Bu tip analiz çekirdeğin küresel mimari gibi nükleer boşluk içinde belirli lokus ve genomik bölgenin davranışının fikir verir. Immunofluorescence, diğer taraftan, nükleer proteinlerin tespiti (modifiye edilmiş histon, histon türevleri ve değiştiriciler, transkripsiyon faktörleri ve makine, nükleer alt-bölme, vb) izin verir. Immünofloresan ve 3B DNA FISH birleştirme içinde önemli bir sorun çekirdeğin 3D mimarisi yanı sıra antikor tarafından tespit edilen epitopu korumak için bir taraftan, ve diğer taraftan, DNA prob nüfuz tespit etmesi için gen lokus veya kromozom bölgeleri 1-5. Burada 3D korunmuş çekirdeklerinde genomik lokus ile kromatin modifikasyonları görselleştirme birleştiren bir protokol sağlar.

DNA ve immüno-FISH B ve T lenfosit gelişimi sırasında antijen reseptörünün bir lokus, V (D) J rekombinasyon işlemi ile bağlantılı olarak nükleer organizasyon değişiklikleri çalışması için Skok laboratuvar kullanılmaktadır. Yukarıda ayrıntılı teknikleri) bize i odağının iki ucu (kasılma) arasında) mesafeleri ölçmek için ii) allel veya lokus (eşleştirme) arasındaki mesafeleri ölçmek, iii), lokus içindeki iv meydana DNA hasarı analiz etkinleştirmek allellerin yerini değerlendirmek ...

Watch this Scientific Journal Video about Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization at JoVE.com

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Burada 3D mikroskopi ve analizler (3D immuno-DNA FISH) izledi DNA FISH immünofloresan ve DNA dizileri tarafından histon modifikasyonları eşzamanlı tespiti için bir protokol açıklar.

3D Nükleer teşkilatında değişiklikler Eğitim için 3D-korunmuş Tane Çekirdekle ilgili Kombine İmmünofloresan ve DNA FISH

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

combined-immunofluorescence-dna-fish-on-3d-preserved-interphase

Research

JoVE Journal

Biology

18.2K Görüntüleme.  New York University School of Medicine. Burada 3D mikroskopi ve analizler (3D immuno-DNA FISH) izledi DNA FISH immünofloresan ve DNA dizileri tarafından histon modifikasyonları eşzamanlı tespiti için bir protokol açıklar.

Video: Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biyoloji

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

DNA tabanlı Balık Türleri Tanımlama Protokolü

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Doğrudan Etiketli Problar kullanarak Sağlam 3D DNA BALIK

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

Bıldırcın-ördek Chimeras kullanma Kraniyofasiyal Kalkınma için Türlere özgü Katılımlar değerlendirilmesi

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Kombine DNA-RNA Floresan<em&gt; In situ</em&gt; Hibridizasyon (FISH) Farklılaştırılmış Kadın Fare Embriyonik Kök Hücreleri X kromozomu inaktivasyonu Eğitim için

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Kronik sonra fare dalak DNA hasar ve Onarım Ölçme<em&gt; In Vivo</em&gt; Beta ve gamma-Radyasyonun Çok Düşük Doz Maruz Kalma

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Belirli Genomik Bölgeler ve enChIP tarafından İlişkili Moleküllerin Tanımlanması İzolasyonu

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Yöntemleri ile Caenorhabditis elegans çağda doğal Protein toplama çalışma değişikliklere

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Cryosec, zebrafish embriyo üzerinde sıralı Immünofluorescence ve Immünhistokimya

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.