Bu çalışma ve ShanghaiTech Üniversitesi, Shanghai, Çin (DP1CA174426 Carlos F. Barbas için) (JL) Ulusal Sağlık Enstitüleri tarafından desteklenmiştir. Moleküler grafik Pymol kullanılarak elde edilmiştir.

1. Klonlama <ol><li> PET-28 ifade vektörü sistemi içine alt-klonlandığında, istek (pET-2F-ZiF) üzerine mevcuttur edilmiştir alanin ile ikame edilmiş, iki parmak ZiF etki elde edilir. 34 </li><li> PCR primerleri ile plazmid zümrüt-pBAD gelen EmGFP amplifiye 5 &#39;Xmal-EmGFP (5&#39;-GGAAATTG CCCGGG ATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3&#39;; kalın olarak Xma I bölgesi) ve 3 &#39;Sacl-EmGFP (5&#39;-CGGATCT GAGCTC TTACTTGTACAGCTCGTCCATGCCGAG-3&#39; ; kalın Sac sitesi). <ol><li> Şablon DNA 5 ng, 10x polimeraz tamponu 10 ul damıtılmış / iyonu giderilmiş su oluşur 1 Taq DNA polimeraz, 0.2 mM her bir dNTP Birimi (U) ve geri kalan hacim 100 ul çözelti içinde, her bir primerden 0.2 uM kullanın . Siklusları: 5 dakika boyunca 95 ° C; 30 saniye için 95 ° C 30 döngü, 30 saniye için 55 ° C sıcaklıkta ve 1 dk için 72 ° C; 10 dakika boyunca 72 ° C. Jel extracti PCR ürünü saflaştırmakve Abs 260 x 50 ug / ml ölçen bir spektrofotometre kullanılarak DNA konsantrasyonunu belirlemek. </li></ol></li><li> Özet pET-2F-ZiF ve sınırlama EmGFP kodlayan uç DNA 1 ug başına enzimin 10 u kullanarak 37 ° C &#39;de 3 saat boyunca tavsiye edilen tamponda Xma I ve Sac I enzimleri. Gibi etidyum bromür gibi floresan interkalasyon boya kullanılarak agaroz jel elektroforezi ile DNA görselleştirmek. </li><li> Jel ekstraksiyon kiti ile sindirilmiş DNA arındırın ve ABS 260 x 50 ug / ml ölçen bir spektrofotometre ile DNA konsantrasyonu belirlenir. </li><li> Oda sıcaklığında en az 1 saat boyunca T4 DNA ligazı 1 ünite kullanılarak pET-2F-ZiF 50-100 ng saflaştırılmış EmGFP kodlayan DNA Arter. 1 insert-vektör molar oranı: En iyi sonuç için, 6 kullanarak ligasyon reaksiyonu gerçekleştirmek. </li><li> Buz üzerinde herhangi bir kimyasal uyumlu XL-1 Mavi E. coli hücreleri 50 ul çözülme ve 10-20 ng lige pET-2F-ZiF-EmGFP bölgesinin ile yavaşça karıştırın. </li><li> 3 için buz tutun0 dak. Isı şoku 90 sn için 42 ° C sıcaklıkta karışım ve çalkalanarak 37 ° C&#39;de 1 saat boyunca katabolik baskı (SOC) ile süper optimal suyu 2 ml hücreler geri kazınılmıştır. </li><li> 50 ug / ml kanamisin içeren bir lizojeni suyu (LB) agar plakası üzerine geri kültür, 100 ul yayıldı ve 37 ° C &#39;de O / N inkübe edin. </li><li> Bir sonraki gün, Süper Broth (SB), ya da 50 ug / ml kanamisin, 37 ° C&#39;de LB agar plakası ve kültür göre O / N itibaren bir koloni içeren LB kültür 6 ml inoküle. Not: Koloni PCR primerlerini 5 &#39;Xmal-EmGFP ve 3&#39; Sacl-EmGFP kullanılarak miniprep önce pozitif klonları tanımlamak için kullanılabilir. </li><li> Miniprep ile pET-2F-ZiF-EmGFP arındırmak ve T7 promotörü kullanılarak DNA dizilemesi ile teyit plazmid (5&#39;-TAATACGACTCACTATAGGG-3 &#39;). </li></ol> 2. İfade ve saflaştırma <ol><li> Kimyasal yetkili BL21 E. 50 ul çözülme Buz üzerinde coli hücreleri ve PL pET-2F-ZiF-EmGFP 100 ng ile yavaşça karıştırınasmid. Adımlarda 1.7-1.8 açıklandığı gibi Transform. </li><li> Ertesi gün, tek bir koloni ile 50 ug / ml kanamisin içeren LB ortamında 10 ml aşılamak ve çalkalanarak 37 ° C&#39;de O / N büyür. </li><li> Bir sonraki gün, 50 ug / ml kanamisin,% 0.2 glikoz ve 100 uM ZnCI2 ile takviye edilmiş LB ortamı 1 L O / N kültür 10 ml seyreltik. 0.8, 600 nm (OD 600) de optik bir yoğunluğa kadar çalkalanarak 37 ° C&#39;de kültür büyütün ve 2 mM izopropil-β-D-1-tiyogalaktopiranosid (IPTG) ve protein ifadesini teşvik eder. 4 ° C&#39;de 10 dakika boyunca 5000 x g&#39;de santrifüj ile 37 ° C&#39;de büyüme 6 saat, hasat sonrasında, hücreler. Not: İndüksiyon koşulları oldukça değişkendir ve ifade edilen proteinlerin istikrar bağlıdır. 0.8 600 ulaşıldığında bir OD kadar OD 600 her 30 dk izleyin. </li><li> Liziz tamponu, 5 ml (50 mM Tris-HCI, 500 mM NaCl içinde hücre pelletini, 100 uM ZnCl2, 1 mM ditiyotreitol (DTT), 1mM MgCl2, 1 mM fenilmetilsülfonil florür (PMSF) ve 10 mM imidazol, pH 8.0). Aşağıdaki ayarı ile sonikasyon ile buz üzerinde lizleyin hücreleri: 50% güç çıkışı, / 10 saniye kapalı aralıklarla üzerinde 5 saniye ile 4 dakika işlem süresi. Çözüm aşırı ısınma kaçının. </li><li> 4 ° C&#39;de 30 dakika boyunca 25,000 x g&#39;de hücre lizat santrifüj ve yeni bir toplama tüpüne süpernatant aktarın. En iyi sonuçlar için, 4 ° C&#39;de aşağıdaki adımları uygulayın. </li><li> Bir sütun dengelenmiş Ni-NTA bulamacın 1 ml kullanıma hazır süpernatanın çalıştırın. Yıkama tamponu, 20 ml (50 mM Tris-HCI, 500 mM NaCl, 100 uM ZnCI2, 1 mM DTT, 1 mM MgCl2, 30 mM imidazol, pH 8.0) ile reçinenin yıkayın. </li><li> 5 yıkama tampon maddesinin ml&#39;si (50 mM Tris-HCI, 500 mM NaCI, 100 uM ZnCI2, 1 mM DTT, 1 mM MgCl2, 300 mM imidazol, pH 8.0) ile proteininin elüt edilmesi. </li><li> Tampon saklama tamponu ile elüt protein (50 mM alışverişiTris-HCI, 500 mM NaCI, 100 uM ZnCl2, 1 mM DTT, 1 mM MgCl2 ve% 10 gliserol, pH 8.0) kullanılarak ve imalatçının talimatlarına uygun olarak santrifüj ile bir spin konsantratör kullanılarak en az 40 uM proteinin konsantre edilir. </li><li> BCA veya Bradford deneyi ile protein konsantrasyonu belirleyin. 10 dakika boyunca 95 ° C &#39;de 2 ul 2 x SDS-PAGE yükleme boyası, kaynatma ile saflaştırılmış proteinlerin 2 ul karıştırın ve daha sonra% 4 protein saflık (Şekil 2) üzere% 20 Tris-Glisin, SDS-PAGE gidermek. </li></ol> 3. Protein Depolama <ol><li> -80 ° C&#39;de, sıvı azotun ve depoda konsantre protein, flaş dondurma alikosu. EmGFP füzyon proteinleri için, ışıktan proteini korumak için alüminyum folyo ile tüp kapsamaktadır. </li><li> Tekrarlanan donma ve protein çözeltisi çözülme kaçının. Not: ZiF-kaynaşık proteinler en az 1 ay boyunca bu koşullar altında stabildir. Uygunsuz veya&gt; 4 ay depolama yol açabilirProtein yağış veya EmGFP ve photobleaching. </li></ol> 4. Protein İletimi <ol><li> % 10 ihtiva eden Dulbecco tadil edilmiş Eagle ortamı (DMEM) içinde HeLa hücrelerini muhafaza (h / h) cenin sığır serumu (FBS) ve% 1 antibiyotik-antimikotik% 5 CO2 ile bir tam nemli bir atmosferde 37 ° C &#39;de. Not: Hücreler fazla 30 kat protein iletimi için tavsiye edilmez pasajlandı. </li><li> Ön kaplama, 25 ° C&#39;de 30 dakika ila 60 poli-lisin, 50 mg / ml 500 ul, 24 oyuklu bir plaka. Oyuk başına 2 x 10 5 hücre yoğunluğunda 24 kuyulu bir levha üzerine tohum hücreleri. Hücreler% 80 arasında -90% konfluent sonra tohumlama 24 saat sonra ya da her bir gözden ortamını çıkarın ve önceden ısıtılmış serum içermeyen DMEM içinde 500 ul (SFM) ile yıkayın. </li><li> Her bir oyuğa, SFM, 250 ul ZiF-EmGFP proteininin 2 uM ve 100 uM ZnCl2 ihtiva eden ekleyin. 1 saat boyunca 37 ° C&#39;de inkübe hücreleri. Not: ZiF etki hücrelerine girmek primarily bir enerji-bağımlı bir mekanizma makropinositozlar, 34 ile. Bu nedenle, hücreleri verimli bir protein içselleştirme 37 ° C&#39;de inkübe edilmesi gerekir. </li><li> Kalsiyum ve magnezyum içermeyen 500 ul ile üç kez hücrelerden Ortamı çıkarın ve yıkama Dulbecco fosfat-tamponlu tuzlu su, heparin, 0.5 mg / ml ile takviye edilmiş (DPBS). Not: Heparin, mansap analizler zorlaştırıyor olabilir yüzey bağlı protein kaldırmak için gereklidir. </li><li> Gelişmiş teslimat için üç kata kadar (İsteğe bağlı) Tekrar tedavileri. </li><li> Hemen 2 dakika süreyle 37 ° C&#39;de% 0.05 tripsin-EDTA ile sindirme ile yıkama heparin sonra muamele edilmiş hücreler izole edin. % 1 FBS ile takviye edilmiş DPBS 0.5 ml müstakil hücreleri yeniden askıya. </li><li> Fluoresein izotiyosiyanat (FITC) kanal (Şekil 3) kullanılarak 35 akış sitometrisi ile her bir numunenin floresan yoğunluğu ölçülür. Nüfusu yerleştirmek için ileri dağılım (FSC) ve yan dağılım (SSC) ayarlayınFarklı özelliklere sahip popülasyonlar birbirinden çözümlenir sağlamak ölçekte faiz. </li><li> Her numune için 10,000 canlı etkinliklerle toplayın ve sadece SOY ile tedavi edilen hücrelere ZiF-EmGFP ile tedavi HeLa hücrelerinde veri analiz yazılımı. 36 Normale floresan flow sitometri kullanarak verileri analiz. </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+flow+cytometry.">Basics of flow cytometry.</a> Methods Mol. Biol. 91, 1-24 (1998).</li><li>Mercer, A. C., Gaj, T., Sirk, S. J., Lamb, B. M., Barbas, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+endogenous+human+gene+expression+by+ligand-inducible+TALE+transcription+factors.">Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.</a> ACS Synth. Biol. 3, 723-730 (2014).</li><li>Segal, D. J., Crotty, J. W., Bhakta, M. S., Barbas, C. F., Horton, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Aart,+a+designed+six-finger+zinc+finger+peptide,+bound+to+DNA.">Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA.</a> J. Mol. Biol. 363, 405-421 (2006).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Burada, hücre-geçirgen çinko-parmağı (ZiF) sahaları kullanılarak protein temini için adım adım bir protokol verilmektedir. ZiF etki kaynaşık enzimatik yük 34 etkinliğini azaltmaz; üretim ve değişikliğe uğratılmamış proteinle birlikte gözlenenler ile hemen hemen aynı mayalarda proteinlerin saflaştırılması için izin verir; ve geleneksel hücre nüfuz peptid ya da protein transdüksiyon alan sistemleri aşan randımanı olan hücre tiplerinin geniş bir aralığı içine proteinler v...

Yüksek verimli ve çok yönlü bir protein teslim stratejileri birçok temel araştırma ve terapötik uygulamalar için kritik öneme sahiptir. hücrelere saflaştırılmış proteinler direkt verilmesi, bunun başarılması için güvenli ve kolay yöntemlerden biri. 1,2 nükleik asitlerin gen ifadesi üzerindeki kullanan stratejiler farklı temsil eder 3-5 proteini dağıtım girmeyle mutagenez riski teşkil bağımsızdır Hücresel transkripsiyon / çeviri makine ve derhal yürürlüğe sağlar. Ancak, proteinlerin üzerine hücre delici aktivitesini endowing basit ve genellenebilir yöntemlerin eksikliği rutin hücre içine doğrudan giriş boşa. Hücre içi protein sağlanmasını kolaylaştırmak için geçerli yöntem, doğal olarak, 6-8 ya da tasarlanan hücre-delici peptidler, 9-12 kompresörlü transdüksiyon etki, 13,14 nano-tanecikleri 15 ve lipozomlar, 16 virüs benzeri partiküller 17,18 meydana kullanımına dayanmaktadır </sup&gt; ve polimerik malzemeler Mikroküre. 19 Ne yazık ki, bu yaklaşımların çoğu düşük hücresel alım oranları, 20,21 yoksul kararlılık, 22 yanlışlıkla hücre tipi özgüllük, 23 düşük endozomal kaçış özellikleri 24 ve ek toksisite. 25 tarafından engellenmektedir, birçok protein iletim teknolojileri teslim proteinlerin biyoaktivitesini azaltır. 14 Biz daha önce laboratuarımızda çinko-parmak nükleazı göstermiştir (ZFN) protein - kimerik kısıtlama programlanabilir Cys 2 -His 2 çinko parmak, DNA bağlama proteini ve Fokl sınırlama endonükleazı 26-28 bölünme alanından oluşan endonükleazlar - doğal olarak hücreye olan geçirgen. 29 Bu şaşırtıcı hücre delici etkinlik özel tasarlanmış çinko-parmak etki, hedeflenen genom tr için güçlü bir araç olarak ortaya çıkmıştır bir DNA-bağlanma platformun bir içsel özelliği olduğu gösterilmiştirMühendisliði, 30-32 ve kabul protein yüzey altı pozitif yüklü kalıntıların takımyıldızı sonucu olduğu. Gerçekten de, c-Jun N-DEK dahil olmak üzere birçok DNA-bağlama proteinleri,., Hücre zarlarını geçme yeteneği doğuştan gelen bir kapasiteye sahip olduğu gösterilmiştir 33 Daha yakın zamanda, laboratuar Bu sonuçlar genişletilmiş göstermiştir ki, çinko-hücre-delici aktivitesi parmak (ZiF) etki hücre içi protein teslimat için kaldıraçlı olabilir. Bir çok geleneksel hücre nüfuz peptit uygulama sistemlerinin aşıldı verimliliği artışa neden oldu spesifik protein yük için bir ya da iki parmak ZiF etki ya da genetik füzyonu. 34 En önemlisi, ZiF aracılı teslim kaynaşık enzimatik kargo aktiviteyi tehlikeye ve kolaylaştırdı Sitozolik teslimat yüksek düzeyde. Topluca, bu bulgular proteinlerin verimli ve kolay teslim kolaylaştırmak için ZiF etki potansiyelini göstermek ve makro potansiyel olarak daha farklı tiplerihücrelere molekülleri. Burada, memeli hücrelerinde protein temini için ZiF teknolojisi nasıl uygulanacağına ilişkin ayrıntılı adım adım bir protokol verilmektedir. Daha önce bir paketi inşa bir, iki, üç, dört, beş ve altı parmak bağlı α-sarmal DNA bağlayıcı artıkların herbirinin, bir ikame, DNA bağlama yeteneğinden yoksun ZiF etki, ancak hücreler 34 (Şekil 1) içine proteinleri üretmektedirler. İki parmak ZiF etki alanı kullanılarak HeLa hücrelerine Emerald GFP (EmGFP) üretimi ve transdüksiyon tarif edilmektedir. Bu protokol, Escherichia coli içinde çözünür ifade ve hemen hemen her memeli hücre tipinde edebilen hemen hemen herhangi bir protein verecek şekilde uzatılabilir. Beklenen sonuçlar sağlanır ve bu sistemin performansını maksimize etmek için stratejiler de tartışılmıştır.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>XmaI</td>
			<td>New England Biolabs</td>
			<td>R0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156L</td>
			<td></td>
		</tr>
		<tr>
			<td>Expand High Fidelity PCR system</td>
			<td>Roche</td>
			<td>11759078001</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini protein gels, 1.5 mm, 10 wells</td>
			<td>Life Technologies</td>
			<td>EC6028BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Life Technologies</td>
			<td>15224-017</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2527I</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Scientific</td>
			<td>R0391</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher Scientific</td>
			<td>PR-V3151&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Thermo Scientific</td>
			<td>36978</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose Resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Units</td>
			<td>EMO Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11966-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic&#160;</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Flat Bottom Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, No Calcium, No Magnesium</td>
			<td>Life Technologies</td>
			<td>21600010</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparan Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>H4777</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop ND-1000 spectrophotometer&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct protein delivery to mammalian cells using cell-permeable cys2-his2 zinc-finger domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

İki parmak ZiF-EmGFP füzyon proteinleri E. ifade edilebilir E. coli,&gt;% 95 homojenlik ve yüksek verimle (&gt; 25 mg / ml) (Şekil 2). Genel bir ve iki parmak ZiF füzyon proteinleri, vahşi tip uğratılmamış proteinin kişilerce hemen hemen aynı miktarlarda üretilebilir. Bununla birlikte, bazı bağlamlarda, beş ve altı parmak ZiF füzyon proteinleri alt baş uygulamalar için yeterince yüksek verimlerle üretilmesi mümkün değildir. 37 ° C&...

Watch this Scientific Journal Video about Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains at JoVE.com

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Çinko-Parmak etki doğal olarak hücre-geçirgen ve memeli hücre türlerinin geniş bir aralığı içine protein verilmesinin aracılık edebilmektedir. Burada, hücre içi protein teslimat için çinko-parmak teknolojisi uygulanması için ayrıntılı bir adım-adım protokolü sunulmuştur.

Hücre geçirgen Cys kullanılarak memeli hücrelerinde doğrudan protein teslim<sub&gt; 2</sub&gt; His<sub&gt; 2</sub&gt; Çinko-parmak Alanlar

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

direct-protein-delivery-to-mammalian-cells-using-cell-permeable-cys2

Research

JoVE Journal

Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

9.8K Görüntüleme.  The Scripps Research Institute.  Çinko-Parmak etki doğal olarak hücre-geçirgen ve memeli hücre türlerinin geniş bir aralığı içine protein verilmesinin aracılık edebilmektedir. Burada, hücre içi protein teslimat için çinko-parmak teknolojisi uygulanması için ayrıntılı bir adım-adım protokolü sunulmuştur. 

Video: Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Geniş Alan Mikroskop kullanarak Uzun vadeli Görüntüleme Memeli Hücreleri

Biyoloji

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Memeli Hücreleri Trypsinizing ve alt kültüre

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

Mühendislik Hücre geçirgen Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

CompoZr Özel Çinko Parmak Nükleazlar ile Genom Düzenleme (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Biyoparlaklık Rezonans Enerji Transferi kullanma Canlı Hücrelerde Protein-protein etkileşimleri araştıran

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Kültür Memeli Hücrelerde Biyolojik tanımında ubikitin ve Ubiquitin benzeri Sistemi Regülatörleri tanımak siRNA Eleme

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

SILAC-immunoprecipitation Kantitatif PROTEOMİK kullanma Memeli Hücrelerde Protein Etkileşim Ortakların Kimlik

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

İnsan Embriyonik Kök Hücreleri Hedefleme Çinko parmak Nükleazlar Geliştirilmiş Gen

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

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.