Orijinal GFP-Pem1 Dr Lei Li bir hediye oldu. Bu çalışma, Ulusal Sağlık Enstitüsü ve Ellison Tıp Vakfı VG hibe tarafından desteklenen ve AS

Bu protokol DSBs geçici ifade nadir bir kesim endonükleaz I-SCEI 3 in vivo olarak indüklenen 1,2 yapıları kromozomal olarak entegre muhabiri ile DNA DSB onarım analizi için bir yöntem tarif. Entegre test DSB kromozomal kapsamında onarım analiz avantaj sağlar. Ancak, bu protokol birincil hücreleri ile çalışırken sorunlu olabilir Pasajlanması uzun süreli hücre, gerektirir. Alternatif bir yaklaşım muhabiri yapıları I-SCEI veya HindIII endonükleaz ile in vitro sindirilir ve daha sonra lineer DNA hücrelerine transfekte ekstrakromozomal testtir . Exctrachromosomal tahlil protokol 4 aşağıdaki değişiklikler aşağıda açıklanan entegre tahlil benzer. Ekstrakromozomal tahlil entegrasyon muhabiri yapıları ihmal ve hücrelerin yerine I-SCEI veya HindIII ve transfeksiyon kontrolü olarak pDsRed2-N1 0.1 mikrogram ile in vitro lineerleştirilmiş plazmid NHEJ muhabiri 0.5 mikrogram ile co-transfect için. İK testi için plazmid ortak transfect 2 mikrogram İK muhabiri I-SCEI veya HindIII ve 0.1 mg pDsRed2-N1 ile lineerleştirilmiş. Transfeksiyon sonra FACS üç gün hücrelerin analiz edin. Ekstrakromozomal testi Pasajlanması geniş hücre kaçınarak, ve birden fazla hücre hatları analizi kolaylaştırmak, analiz için birincil izolatlarında DSB onarım sağlar. 1. Muhabir oluşturur NHEJ ve İK NHEJ muhabiri kaset 5 Pem1 gen (GFP-Pem1) bir mühendislik 3 kb intron ile GFP geninin içerir. Pem1 intron DSBs indüksiyonu için HindIII ve I-SCEI endonükleaz (Şekil 1a) tanınması dizileri ile çevrili bir adenoviral ekzon içerir. I-SCEI siteleri ters yönde. I-SCEI nonpalindromic bir tanıma dizisi, dolayısıyla iki ters siteleri uyumsuz DNA biter (Şekil 1c) üretir. Uyumsuz DNA en iyi doğal olarak meydana gelen DSBs taklit sona erer. Sağlam NHEJ kaset adenoviral ekson GFP ORF bozar gibi negatif GFP. I-SCEI DSBs indüksiyonu üzerine, adenoviral ekson kaldırılır ve NHEJ GFP geninin fonksiyonu (Şekil 2) geri yükler. Bu NHEJ muhabiri benzersiz özellik intron silme ve eklemeler tolere edebilir NHEJ olayların geniş bir yelpazede algılayabilir. İK muhabiri kaset 1 (Şekil 1b), GFP-Pem1 dayanmaktadır . İK kaset, GFP-Pem1 ilk ekson üç kısıtlama siteleri, I-SceI/HindIII/I-SceI ekleme ile birlikte 22 bp silinmesini içeriyor. Silinmesi, GFP bir NHEJ olay tarafından sulandırılmış olamaz sağlar. Iki I-SCEI siteleri böylece I-SCEI sindirim uyumsuz uçları bırakır (Şekil 1c), ters yönde. GFP-Pem1 ilk kopyası GFP-Pem1 promoter-less/ATG-less ilk ekzon ve intron tarafından takip edilmektedir. Sağlam inşa GFP-negatif. I-SCEI sindirim DSB indüksiyon üzerine fonksiyonel GFP genini ilk GFP-Pem1 ekson mutasyona uğramış iki kopya arasında molekül veya moleküller arası gen dönüşümü ile sulandırılmış. GFP geninin ikinci kopya, ilk ATG kodon ve ikinci ekson eksik gecicek ya da tek iplikli tavlama olduğundan GFP faaliyeti geri olmayacaktır. Bu tasarım, memeli hücrelerinde baskın İK yolu (Şekil 3) gen dönüşüm özel algılama, sağlar. 2. Muhabir Entegrasyonu Genom içine oluşturur. <ol><li> NHEJ veya İK muhabiri plazmid yüksek kalitede bir hazırlık olun. En iyi sonuçlar Qiagen Endo Ücretsiz Kiti kullanımı içindir. 260/280 nm&#39;de absorbans plazmid konsantrasyonu ve saflık ölçün. </li><li> 50 ul NheI (reaksiyon toplam hacmi 50 U) 6 saat 37 ile sindirerek plazmid muhabiri 10 mikrogram Linearize ° C inkübatör (buharlaşmasını önlemek için, kuru blok kullanmayın). Qiagen QiaexII Takımı, 10 mM Tris-HCl pH: 8.0 20 ul içine Zehir DNA sindirimi çözüm DNA arındırın. 260/280 nm&#39;de absorbans DNA konsantrasyonu ve saflık ölçün. Genellikle, arıtma sırasında DNA&#39;nın% 20-30 kaybedersiniz. Sindirilmemiş bir muhabir inşa ile birlikte bir jel üzerinde küçük bir kısım (2 ul) çalışan verim ve sindirim, onaylayın. Saflaştırılmış Lineerleştirilmiş muhabiri yapı 20 ° C&#39;de saklanabilir. </li><li> Transfeksiyon hazırlık, hücrelerin en iyi büyüyen durumuna getirmek. Eğer donmuş bir şişeden başlangıç, transfeksiyon önce iki kez hücrelere bölünmüş. Konfluent hücrelerinin bir tabak başlayarak, bir kez ayrıldı. </li><li> Hücrelerin% 70-80 izdiham (genellikle normal insan fibroblastlar için iki gün, eğer kaplama 5x10 5 cells/100 mm levha) büyütün. Bu logaritmik büyüme hücreleri getirmek için en iyi transfeksiyon verimlilik için kritik önem taşımaktadır. M aşamasında aktif olarak büyüyen kültür hücreleri (yuvarlak hücreler yüzeye bağlı olarak görülen) içerir. </li><li> TransfAmaxa Nucleofector aşağıdaki üreticinin tavsiyelerini kullanarak hücreleri vb. Fibroblastlar NHDF çözüm ve programı U20 kullanım içindir. Transfeksiyon için, iki adet 100 mm plakalar (~ 2x10 6 hücreleri) hücreler hasat . Bu hücrelerin aşırı trypsinize için kritik öneme sahiptir. Plakadan müstakil hücrelerinin% 80 olarak en kısa sürede trypsinization durdurun. NHDF çözüm hücrelerin yeniden süspanse edin. Hücreler NHDF çözüm duyarlıdır, bu nedenle, kuluçka süresi en aza indirmek ve hücreleri hafifçe karıştırın. Lineerleştirilmiş muhabiri inşa 0.5 mikrogram ile hücreleri Transfect. Normal insan fibroblastlar sonucu bir muhabir tek bir kopyasını entegrasyonu ile kullanılan bu transfeksiyon koşulları, integrants çoğunluğu oluştururlar. </li><li> 1 mg / ml geneticin (G418) 24 saat sonra transfeksiyon ekleyerek kromozomal olarak entegre muhabiri yapıları hücreleri seçin. 7-10 gün için seçim devam edin. </li><li> G418 dirençli koloniler (tek tek koloniler veya havuz dirençli klonlar seçebilirsiniz) seçin. Yaklaşık beş adet 100 mm plakalar kültür genişletin. Ileride kullanmak için üç tabak dondurma ve istenilen hücre sayısı (genellikle on adet 100 mm plakalar beş transfections için yeterli) kalan iki tabak genişletmek. </li><li> Hücrelerin% 70-80 izdiham (100 mm fibroblastlar için plaka başına 5x10 5 hücre kaplama iki gün sonra) ulaştığında, hücreler I-SCEI plazmid ifade ile transfekte hazır. </li></ol> 3. I-SCEI Endonuclease geçici İfade DSB İndüksiyon <ol><li> Transfect Amaxa Nucleofector (fibroblastlar için NHDF çözüm ve U20 programı kullanarak bir transfeksiyon verimlilik kontrolü gibi ifade plazmid ve 0.1 mg pDsRed2-N1 (Clontech) 5 mg I-SCEI karışımı ile logaritmik olarak büyüyen kültür ~ 2x10 6 hücre (iki tabak) .) </li><li> (Ii) 5 mg pDsRed2-N1; bir kontrol (iii) 5 mg plazmid bir ifade değildir bu, aynı zamanda 5 mg EGFP ifade plazmid (i) ~ 2x10 6 hücre trasfecting FACS üç kalibrasyon kontrolleri hazırlamak floresan protein (plazmid kodlama HPRT minigene kullanıyorum). </li><li> GFP ve DsRed proteinleri ifade için yeterli zaman sağlamak için dört gün boyunca hücreler büyürler. </li><li> Transfeksiyon sonra, inverted mikroskop, GFP ve DsRed tespiti için filtreler ile floresan transfeksiyon ve GFP ve DsRed proteinleri ifade etkinliğini doğrulamak (isteğe bağlı) Üç gün. </li></ol> 4. Akış Sitometri GFP + ve DsRed + Hücreler Analizi <ol><li> Dört gün transfeksiyon hasat I-SCEI transfekte hücreleri ve kontrol hücreleri. PBS 500 mcL hücrelerin yeniden süspanse ve FACS tüpleri hücreleri aktarmak. Bu aşamada tüm hücreleri hasat ve yuvarlak hücreler için çok önemlidir. Bunu başarmak için, bu, uzun bir trypsinization süre ya da daha güçlü bir tripsin kullanılması tavsiye edilir. Hücrelerin% 99 plaka kopuk olduğunu onaylayın ve yuvarlak hücrelerin mikroskop altında gözlemleyerek var. (Folyo ile buz kovasının kapağını), ışıktan koruyarak buz üzerinde hücreleri saklayınız. Bir kaç saat buz üzerinde hücreleri saklamak mümkündür. En iyi sonuçları hemen hasattan sonra hücrelerin analiz. </li><li> GFP, DsRed ve negatif kontroller FACS Kalibre. Analizinde tüm floresan hücreleri (Şekil 4) dahil etmek için gerilim ve renk tazminat ayarlayın. Deneysel örneklerin floresan yoğunluğu daha kontrollerin düşük olacağını aklınızda bulundurun. </li><li> Analiz I-SCEI FACS transfekte hücreler. Her tedavi için en az 20.000 hücreleri saymak. GFP ve DsRed floresan GFP yüzde + veya DsRed + hücrelerin% 25 altında tutulması tavsiye edilir. Arasındaki potansiyel bir girişimi önlemek için DsRed + veya GFP + hücrelerin yüzdesi yüksek ise pDsRed2-N1 veya I-SCEI plazmid miktarını azaltır. Deneylerde sadece plazmid pDsRed2-N1 miktarını ayarlamak için gerekli değil, I-SCEI plazmid. </li><li> GFP + hücreler, DNA DSB onarım verimliliği yüzde karşılık gelir ve DsRed + hücrelerinin yüzde transfeksiyon etkinliğini gösterir. DsRed + hücreleri GFP + hücrelerin oranı göreli olarak DNA DSB onarım verimliliği hesaplayın. </li><li> Tamir sonuna kavşak onarım doğruluğunu test etmek için sıralı olabilir. Muhabir yapıları, çoğaltma E.coli kökeni ve kanamisin direnç genleri (Şekil 1) içerir . Bu, genomik DNA EcoRI enzim, recircularization ve yetkili E.coli hücreleri dönüşüm ile sindirerek yapıları tahlisiye sağlar . Kurtarıldı plazmid sonra kısıtlama sindirim ve sıralama ile analiz edilir. </li></ol> 5. Temsilcisi Sonuçlar Şekil 4 ve 5 kontrol transfections ve NHEJ ve İK deneyleri Tipik FACS sonuçlar gösterilmiştir. Floresan yoğunluğu ve floresan hücrelerinin sayısı bütünleşik içeren I-SCEI transfekte hücreler daha düşük olacaktıred NHEJ ve İK yapıları (Şekil 5), GFP ve bu hücrelerin DsRed yapıları kopya sayısı farkı nedeniyle denetimleri (Şekil 4) karşılaştırıldı. Deneyde Her hücre entegre muhabiri yapının sadece bir kopyasını içerir, böylece başarılı onarım olaylar sulandırmak hücrelerin bir kısmı, GFP geninin. Insan fibroblastlar 0.1 mikrogram pDsRed2-N1 Transfeksiyon hücre başına yaklaşık 1 plazmid kopyasını sağlar. NHEJ ve İK etkinliğinin GFP + / DsRed + hücreleri bir oranı olarak hesaplanır. NHEJ 2 insan hücrelerinde İK daha verimli bir süreçtir. Insan fibroblastlarında NHEJ verimliliği genellikle 0,6-1,3, ve İK verimliliği 0.05-0.3. DSB verimliliği bir oranı GFP olarak ölçülür + / DsRed + I-SCEI ve DsRed2-N1 plazmid karışım hücreleri varyasyonlar sonucu etkileyebilir. Plazmid kalitesi de transfeksiyon verimliliği değiştirerek sonuçlarını etkileyebilir. Bu nedenle, tutarlı ölçümler elde etmek, bir deneyde içinde aynı plazmid karışımı kullanmak önemlidir. Ayrıca, DSB onarım verimliliği, hücre tipi, hücre döngüsü faz ve entegre yapıları kromozomal konumu etkilenir. <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="Şekil 1" /> Şekil 1. Muhabir NHEJ (A) ve İnsan Kaynakları (B), DNA DSB onarım analizi için oluşturur . (C) Uyumsuz DNA iki ters I-SCEI siteleri sindirim tarafından oluşturulan biter SD, ek donör; SA, ek alıcısı, gölgeli meydanları, polyadenylation siteler; iki yararlanıcı tarafından kontrol Neo / Kana, tek ORF: SV40 görüşmekte neomisin direnç memeli hücreleri; ve E. coli β-laktamaz görüşmekte kanamicyn direnci; Ori, E. Coli puc çoğaltma kökenlidir. <img src="/files/ftp_upload/2002/2002fig2.jpg" alt="Şekil 2" /> Şekil 2. . Muhabir NHEJ analizi için inşa GFP geninin etkin yapı, ancak bir DSB ve başarılı NHEJ indüksiyonu üzerine inşa GFP + olur. Aşağıda gösterildiği NHEJ bu muhabir bir onarım ürün. <img src="/files/ftp_upload/2002/2002fig3.jpg" alt="Şekil 3" /> Şekil 3. Muhabir, gen dönüşümü ile HR analizi için inşa gen dönüşüm (GC) I-SCEI, İK DSBs indüksiyonu sırasında aktif bir GFP genini yeniden . GC, crossing-over (X-over), tek iplikçik tavlama (SSA) ve NHEJ: Aşağıda gösterilen, büyük DNA onarım yolları bu muhabir tamir ürünlerdir. X-over onarım ürün intramoleküler rekombinasyon için gösterilir, exramolecular rekombinasyon aynı ürün, ama bir kromozom üzerinde yer alan olacaktır. Ifade aktif GFP protein genleri (GFP +) yeşil renk ile gösterilir. <img src="/files/ftp_upload/2002/2002fig4.jpg" alt="Şekil 4" /> Şekil 4. FACS analizi için parametreleri Kalibrasyon (A) Fibroblastlar oto floresan hücreleri hariç olmak için bir negatif kontrol olarak kullanılan plazmid pHPRT transfekte. (B) Fibroblastlar GFP + hücreler (yeşil alan) için yolluk ayarı için kullanılan, plazmid pEGFP ile transfekte. (C) Fibroblastlar DsRed + hücreleri (kırmızı bölge) için yolluk ayarı için kullanılan plazmid pDsRed2-N1, transfekte. <img src="/files/ftp_upload/2002/2002fig5.jpg" alt="Şekil 5" /> Şekil 5. Tipik kromozomal olarak entegre muhabiri yapıları ile normal insan fibroblastlarında NHEJ (A) ve İnsan Kaynakları (B) analiz sonuçları.

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

Floresan NHEJ ve İK muhabiri deneyleri, in vivo olarak ayrı ayrı her bir DSB onarım yolu ölçmek için nicel bir yol sağlar. Deneyleri, FACS güvenilir 20.000 hücre 10 GFP + hücreleri tespit edebilen çok duyarlıdır. Testler GFP + hücreler görünümünü DSBs 2 indüksiyon sonrası dakika veya saat içinde tespit ederek &quot;gerçek zamanlı&quot; onarım olaylarını ölçmek için adapte olabilir. Ayrıca, GFP + hücrelerin analizi DSB onarım sonra farklı hücre döngüsü aşamasın...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>EndoFree Plasmid Maxi kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12362</td>
<td>&nbsp;</td>
<tr>
<td>Qiaex II Gel Extraction Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>20021</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa Nucleofector</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
<tr>
<td>Geneticin (G418)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11811-031</td>
<td>&nbsp;</td>
<tr>
<td>pDsRed2-N1</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>632406</td>
<td>&nbsp;</td>
<tr>
<td>Round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352058</td>
<td>FACS tubes</td>
</tr>		</tbody>
		</table>
		</div>

analysis of dna double-strand break (dsb) repair in mammalian cells

DNA çift iplikli sonları büyük kaybı genetik bilgi ve hücre ölümüne yol açabilecek en tehlikeli DNA lezyonlar. Hücreler onarım DSBs katılmanın nonhomologous sonunda (NHEJ) ve homolog rekombinasyon (HR): iki ana yollar kullanarak. NHEJ ve İK pertürbasyonlarının genellikle erken yaşlanma ve tümörogenez ile ilişkili, dolayısıyla her DSB onarım yolu ölçme nicel bir şekilde olması önemlidir. Laboratuvarımız NHEJ ve İK hassas ve kantitatif ölçümüne olanak floresan muhabiri yapıları geliştirmiştir. Yapıları DSBs indüksiyonu için nadir bir kesme-SCEI endonükleaz tanınması siteleri içeren bir mühendislik, GFP geninin dayanmaktadır. Ek bir ekson GFP geninin inaktive veya mutasyonlarla olduğu gibi başlangıç ​​yapıları GFP negatif. NHEJ veya İK I-SCEI indüklenen sonlarının Başarılı onarım fonksiyonel GFP genini geri yükler. Flow sitometri sayılır GFP pozitif hücrelerin sayısı NHEJ kantitatif ölçü veya İK verimliliği sağlar.

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

Bu makale, GFP tabanlı floresan açıklar<em&gt; In vivo</em&gt; Homolog rekombinasyon ayrı ayrı ölçmek ve nonhomologous memeli hücrelerinde katılmadan sonunda testleri.

Memeli Hücrelerde DNA çift iplikli Break (DSB) Tamir Analizi

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

analysis-of-dna-double-strand-break-dsb-repair-in-mammalian-cells

Research

JoVE Journal

Biology

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

31.5K Görüntüleme.  University of Rochester. Bu makale, GFP tabanlı floresan açıklar In vivo Homolog rekombinasyon ayrı ayrı ölçmek ve nonhomologous memeli hücrelerinde katılmadan sonunda testleri.

Video: Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Memeli İskelet Kaslar kullanarak DNA Transfeksiyon In Vivo Elektroporasyon

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Biyoloji

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Yetişkin grubuyla Memeli Nöronlar içine DNA intranükleer Mikroenjeksiyon

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Memeli Hücrelerinde Glikoproteinler N-bağlı Şeker Zincirler Pulse-kovalamaca Analizi

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

İyon Kanalları ve Fonksiyonel Analiz Mutagenez Heterologously Memeli Hücreleri cinsinden ifade edilir

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann-astrosit Etkileşimleri Analizi In Vitro Tahliller

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Mikroenjeksiyon tarafından Memeli Hücreleri mRNA Nükleer İhracat Kinetik Analizi

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Memeli Hücreler, Hücre Döngüsü Pozisyon Analizi

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.