Bu eser kısmen başlangıç Cincinnati çocuk Araştırma Vakfı (CCRF) üzerinden M.N. ve yenilikçi çekirdek Grant (ICG) ulusal sağlık Enstitüleri altında ilerleyen translasyonel Bilimler için Ulusal Merkezi tarafından desteklenen için finansman tarafından desteklenen Ödülü sayı UL1 TR001425. İçeriği yalnızca yazarlar sorumludur ve mutlaka Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmiyor.

Protokolü özetleyen bir örnek için bkz: şekil 1 .
1. Baculovirus plazmid DNA saflaştırılması
<ol><li>Bakteriyel kültür baculoviral plazmid DNA (bacmid)14 200 ml LB suyu antibiyotik içeren büyümek; sefaloridin (50 µg/mL), tetrasiklin (10 µg/mL) ve viomisin (7 µg/mL), orbital Çalkalayıcı ayarını 300 rpm'de 37 ° C'de 16 h için.</li>
	<li>Standart alkalin lizis protokolü15kullanarak bakteriyel kültüründen DNA bacmid arındırmak ve bacmid TE arabelleği geçiyoruz.</li>
</ol>2. Baculovirus imalatı
<ol><li>Kültür Sf9 orbital Çalkalayıcı kuluçka 135 devir/dakika ve 28 ° C'de bir polikarbonat Erlenmeyer şişe serum içermeyen böcek kültür orta1,2,3içeren hücrelerde. Not: Sf9 hücreleri donmuş stoktan çözdürülen hücrelere yeniden elde etmek ve üstel büyüme aşaması girmek için en az iki hafta izin vermesi.</li>
	<li>Üstel büyüme bir hemasitometre ile Trypan mavi ile boyama sonra aşaması, hasat Sf9 hücreleri saymak. 1 x 106 Sf9 hücrelerin 6-iyi doku kültürü tedavi tabakta orta 2 ml bir kuyu başına tohum. Hücre bir kuluçka 28 ° C'de 1 h için eklemek için izin verir.</li>
	<li>Büyüme orta ile 1 mL serum-Alerjik unsupplemented Grace'in böcek kültür orta yerine.</li>
	<li>Böcek hücre transfection reaktif kullanarak Sf9 hücre içine bacmid DNA transfect.<ol>
<li>Mix 8 µL böcek hücre transfection reaktif Grace'in böcek orta 100 µL ile.</li>
		<li>Bacmid DNA unsupplemented böcek orta 100 µL içine 2 µg sulandırmak ve karışımı yavaşça vortexing tarafından.</li>
		<li>Seyreltilmiş DNA seyreltilmiş böcek hücre transfection reaktif ile birleştirmek ve karışımı yavaşça. 15-30 dakika oda sıcaklığında için kuluçkaya.</li>
		<li>Dropwise hücreleri üzerine DNA-lipid karışımı ekleyin. Yaklaşık 5 h için bir kuluçka 28 ° C'de kuluçkaya.</li>
	</ol>
</li>
	<li>Transfection karışımı hücrelerdeki kaldırmak ve PBS 2 mL hücrelerle yıkayın.</li>
	<li>Böcek kültür orta 2 mL ekleyin ve kültür bir kuluçka 28 ° C'de medya değiştirmeden devam ediyor. Not: Bacmid floresan bir protein içeriyorsa, onun ifade hücreleri 48 saat sonrası transfection içinde tespit edilebilir. Baculovirus transfected Sf9 hücreleri tarafından üretilen ve daha sonra untransfected hücreleri bozar kültür orta salgılanan. Tüm hücre nüfus baculovirus ile 5 gün içinde enfekte olur ve cytopathic etkisi olarak da adlandırılan viral enfeksiyon belirtileri gösterir. Bu artan hücre çapı içerir, çarpıtmalar/granül sitoplazma, hücre büyümesini kesilmesi ve ölü ya da lysed hücreleri hücre kültüründe mevcut. Ancak, transfection veya enfeksiyon verimliliği en uygun değilse, kültür açık bir cytopathic etkisi göstermeyebilir. Cytopathic etkisi ile 200-400 X büyütme, bir ters faz mikroskobu görüntülenmeyecektir. Enfekte Baculovirus hücre anti-gp64 antikor11ile boyama tarafından tespit edilebilir.</li>
	<li>Baculovirus süpernatant 5 gün P1 virüs stok olarak transfection ve etiket sonra hasat.</li>
	<li>Karanlıkta % 0.5 sığır serum albumin (BSA) ile ışığa duyarlı olan baculovirus süpernatant, PBS içinde saklayın. Kısa süreli (en fazla 90 gün) için 4 ° C'de veya uzun vadeli için-80 ° C'de depolayın.</li>
	<li>Tohum 6 × 106 Sf9 hücreleri doku kültürü tedavi bir 10 cm tabak içinde böcek kültür ortamının 10 mL. İlk baculovirus süpernatant (P1) 1 mL hücrelere ekleyin.</li>
	<li>Baculovirus süpernatant (2P) 5inci gün sonrası enfeksiyon, hasat.</li>
	<li>Tohum 50 mL süspansiyon kültür 250 mL polikarbonat Erlenmeyer flask böcek kültür ortamında Sf9 hücrelerinde (3 x 106 hücre/mL) ve yer bir orbital Çalkalayıcı kuluçka şişeye. P2 stokunun 2 mL hücrelere ekleme ve 28 ° c sabit sıcaklık koruyarak 135 rpm'de sallamak Not: Üç ya da dört gün sonra enfeksiyon, tüm Sf9 hücre popülasyonu yüksek titresi baculovirus üretim bir göstergesidir cytopathic etkisi gösterir.</li>
	<li>İstediğiniz birimin (P3P4,...) elde edilir kadar sırayla yükseltmek baculovirus supernatants 10 50-fold sonraki her enfeksiyon hücre kültüründe hacmiyle artırarak. Not: P3 3rd yuvarlak enfeksiyon Sf9 1 litre hangi 500 mL içinde hücre kültürü P2 baculovirus süpernatant ile 10-20 mL etkilenmiş olabileceğini belirtir. P4 4th yuvarlak hangi 5-10 L Sf9 hücre kültürü P3 baculovirus süpernatant ve benzeri 100-200 mL ile enfekte enfeksiyon var. Genellikle, Sf9 hücreleri, 3 × 106 hücre/mL serum içermeyen böcek kültür medya seribaşı.</li>
	<li>Baculovirus 30 dk 4 ° C'de hücresel enkaz kaldırma ve süpernatant ile 50 adet/mL nükleaz (250 adet/µL stokunun) için 2 h baculovirus 37 ° C genomik DNA ve RNA sindirmek için tedavi için 1000 x g de süpernatant santrifüj kapasitesi. Supernatants 0,45 mikron filtrasyon ünitesi aracılığıyla filtre.</li>
</ol>3. Kromatografi Sistemi hazırlanması
<ol><li>Sırayla örnek ve arabellek satırları her steril su, 1 N sodyum hidroksit, su ile durulama tarafından Kromatografi Sistemi hazırlamak ve arabellek (20 mM sodyum fosfat tampon içeren 150 mM Sodyum Klorür, pH 7,0) 50 mL/dk bir doğrusal akış hızı yıkayın.</li>
	<li>Arabellek (5 CV steril 20 mM sodyum fosfat tampon içeren 2 M Sodyum Klorür, pH 7,0) temizlik sütun sırayla çalıştırarak heparin 50 µm sütun (10 × 10 cm, 7,9 mL (CV) sütun biriminin) hazırlamak ve 5 CV yıkama tampon 7 mL/dk doğrusal akış hızında.</li>
</ol>4. Baculovirus vektör saflaştırılması
<ol><li>Kromatografi Sistemi aşağıdaki gibi ayarlayın:<ol>
<li>Giriş bağlantı noktası süpernatant baculovirus yüklemek için yükleme örneği kullanmak.</li>
		<li>Arabellek girişi bağlantı noktası A1 yıkama arabellek yüklemek için kullanın. En az 500 mL şişe yıkama arabelleği hazırlamak ve yıkama arabellek satırı şişe içine koyun.</li>
		<li>Arabellek girişi bağlantı noktası A2 elüsyon arabellek (1.5 M Sodyum Klorür, pH 8.0 ile 20 mM sodyum fosfat) yüklemek için kullanın. En az 500 mL şişe elüsyon arabelleği hazırlamak ve elüsyon arabellek satırı şişe içine koyun.</li>
		<li>Birkaç 50 mL konik tüpler süpernatant sütun doğrudan baculovirus toplamak için kesir toplayıcı, yıkama arabellek ve eluted baculovirus yerleştirin.</li>
	</ol>
</li>
	<li>Heparin sütun yıkama arabelleği (40 mL) 7.0 mL/dk doğrusal akış hızında beş 7.9 mL sütun birimler (CVs) ile equilibrate.</li>
	<li>250 mL baculovirus süpernatant Kromatografi Sistemi örnek pompa (giriş örnek bağlantı noktası) 2.0 mL/dak bir doğrusal akış hızında çalıştırarak heparin sütuna yerleştirin.</li>
	<li>10 CVs (80 mL) yıkama arabellek heparin sütun 2.0 mL/dk doğrusal akış hızında ultraviyole (UV) Absorbans eğrisi kadar koşmak-den geçerek (280 nm) satır taban çizgisine geri döndü ve kararlı hale gelir.</li>
	<li>Ne zaman baculovirus parçacıklar heparin sütundan ayırmak elüsyon arabelleği 4.0 mL/dak izle Kromatografik protein keskin elüsyon zirvesine için doğrusal akış hızında baculovirus parçacıklar 7.9 mL heparin sütun 5 CVs (40 mL) ile elute.</li>
	<li>Sonrası elüsyon, hemen 10 kat baculovirus parçacıklar osmotik şoktan inactivation sonraki Santrifüjü sırasında önlemek için suda 20 mM sodyum fosfat tampon kullanarak eluted baculovirus süpernatant sulandırmak.</li>
	<li>Baculovirus süpernatant, yıkama arabellek ve eluate yükleme sırasında toplanan sütun akış yoluyla dahil olmak üzere, her birinin kesirler 100 µL depolamak. HT1080 arıtma işlemi (bakınız Bölüm 6) değerlendirmek için bu baculovirus kesirler bulaştırmak.</li>
	<li>Sütun Temizleme arabellek ve yıkama arabellek sütun ile temizleyin. Son olarak, doğrusal akış 7.0 mL/dk hızında sütun 5 CV steril % 20 etanol su ile durulayın ve 4 ° C'de depolayın</li>
	<li>Su, su ile tekrar 1 N sodyum hidroksit ile % 20 etanol suda 50.0 mL/dk doğrusal akış hızında Kromatografi Sistemi temiz ve sistem % 20 etanol içinde saklayın.</li>
</ol>5. konsantrasyonu Baculovirus
<ol><li>Santrifüj tüpleri bir otoklav kullanarak önceden sterilize. Her ultracentrifuge tüpler doku kültürü başlıklı baculovirus süpernatant eşit bir hacmi ekleyin. Bir denge tarafından tüp ağırlık ölçmek ve steril PBS ultracentrifuge tüplerini içeriğini ağırlığını ayarlamak.</li>
	<li>Her biri ultracentrifuge tüpler kova SW 32 Ti rotor muhalif koyun.</li>
	<li>Ultracentrifuge de 80.000 × g çalıştırmak 4 ° C'de 90 dk</li>
	<li>Ultracentrifuge tüpler doku kültürü mahallede açın ve aseptik baculovirus Pelet kaldırmadan sıvı Aspire edin.</li>
	<li>Her tüpün Pelet şiddetle aşağı yukarı 200 µL % 0,5 (w/vol) BSA içeren PBS ile pipetting tarafından resuspend.</li>
	<li>Cryovials (şişe başına 50-100 µL) için konsantre baculovirus aktarın ve-80 ° C'de depolayın</li>
</ol>6. Baculovirus titrasyon
<ol><li>Enfeksiyon, önceki gün bir hemasitometre Trypan hücrelerle mavi boyama sonra kullanarak HT1080 hücreleri saymak. 6-şey plaka 2 ml, Dulbecco'nın modifiye kartal orta (% 10 fetal sığır serum (FBS) içeren DMEM) bir kuyu başına 1 x 105 HT1080 hücre tohum. Tohum birkaç ek wells hücreleri enfeksiyon anda mevcut tam sayısını belirlemek için. Kuluçka 37 ° C ve % 5 CO2hücrelerinde kültür. Not: Onlar heparan sülfat diğer hücre hatları16ile karşılaştırıldığında daha yüksek bir seviyede hızlı gibi HT1080 hücreler titrasyon için kullanılır. HT1080 hücreleri olduğundan yapışık, titrasyon daha basit ve daha az zaman alıcı ile karşılaştırıldığında geleneksel Sf9 tabanlı titrasyon kullanarak.</li>
	<li>Enfeksiyon günü, iyi başına hücre sayısı hücreleri kuyulardan sayarak belirler. Hücreleri bir kenara önce sütun arıtma ve örnekleri ile sütun akış yoluyla, yıkama ve elüsyon kesirler belirlenmiş olduğunu seyreltilmiş orijinal baculovirus ekleyerek bulaştırmak. Her örnek için seyreltme ve ampirik olarak bulaşıcı baculovirus parçacıkları üzerinde temel birim belirlemek ve her kuyuya nüsha bulaştırmak.</li>
	<li>İki gün sonra enfeksiyon, hücrelerin gen ilgi bir ifade için analiz. Hücreleri floresan protein (örneğin GFP)17ifade edersek bir Akış Sitometresi kullanılarak analiz edilebilir.</li>
	<li>Şimdiki zaman enfeksiyon, seyreltme faktörü ve yüzdeleri formülü kullanarak hücreleri floresan proteinin hücre sayısına titreleri (bulaşıcı birim mL) hesaplamak: (sırasında enfeksiyon × yüzde floresan proteinin hücre sayısı pozitif hücrelerinin × seyreltme faktörü) / baculovirus ml hacmi. Not: Örneğin, hücreleri enfeksiyon zaman kuyuda başına 1,2 × 105toplam sayısıdır, floresan protein oranı % 5'dir, seyreltme faktörü 100 ise seyreltilmiş baculovirus eklendi hacmi 10 µL titresi 6 × 107 bulaşıcı birim (IU) ise, /mL.</li>
</ol>

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Yazarlar hiçbir çıkar çatışması beyan ederim.

Burada sunulan Protokolü baculovirus süspansiyon kültür Sf9 hücrelerde üretimi ve heparin benzeşme Kromatografi kullanarak baculovirus arınma açıklar. Bu protokol için kullanılan parametreler verimi en üst düzeye çıkarmak ve bulaşıcı baculovirus inactivation en aza indirmek. Burada sağlanan iletişim kuralı baculovirus parçacıklar iyileşme ile karşılaştırıldığında önemli ölçüde geliştirilmiş bir kurtarma başkaları tarafından9elde gösterir.
<p class="jo...

Baculovirus öncelikle rekombinant proteinler ve Lepidoptera Spodoptera fugiperda (Sf) 9 aşı üretimi için kullanılan rekombinant Autographa californica multicapsid nükleer polyhedrosis virüs (AcMNPV) kullanarak böcek hücreleri 1 , 2 , 3 , 4. son zamanlarda, gen terapisi uygulama5için umut verici bir vektör olarak ortaya çıkıyor. Bu geniş bir ev sahibi ve doku tropism sahip olduğu bilinmektedir, durgun ve Proliferasyona hücreleri bozar patojenik olmayan ve ana bilgisayar kromozom4,5,6entegre değil. Ayrıca, ölçeklenebilir ve kapalı sistem için gelecekteki klinik üretim1işlemek sağlar süspansiyon serum serbest kültür baculovirus üretilebilir.
Baculovirus parçacıklar saflığı sitotoksisite7,8,9en aza indirirken etkili iletim ulaşmak için önemlidir. Baculovirus ultrasantrifüj veya teğet akışı filtrasyon (TFF) onun infektivite üzerinde sınırlı etkisi ile konsantre. Ancak, bu işlemler sadece virüs parçacıkları konsantre aynı zamanda hücre artıkları ve proteinlerin Sf9 gelen Kültür, hangi-ebilmek var olmak zehirli vitro (kişisel gözlem) ve iltihap veya bir bağışıklık kullanıldığında yanıt neden olabilir vivo içinde. Bu, özellikle yüksek kullanarak virüs stokları konsantre önlemek için bulaşıcı baculovirus saflaştırılmış ve parçacıklar kirletici ayrılmış gerekir.
Birkaç yöntem arıtma ve toplama baculovirus vektörler10,11,12için rapor edilmiştir. Kullanılabilir yaklaşımlar, heparin benzeşme Kromatografi tek adımlı bir üst düzey arıtma proteinler12kirletici düşük konsantrasyon ile sağlar. Yönteminin kimliği heparan sülfat ile reseptör baculovirus13,14dayanmaktadır. Sf9 hücre süpernatant üstüne belgili tanımlık sütun ve baculovirus bağlama yükledikten sonra sütun ilişkisiz veya gevşek bağlı bulaşıcı parçacıkları kaldırmak için fizyolojik (izotonik) arabellek ile yıkanabilir. Heparin bağlama tersinir olduğundan, baculovirus parçacıklar hemen inactivation önlemek için fizyolojik (izotonik) tuz konsantrasyonu için ozmotik şok12ile seyreltilmiş yüksek bir salt arabellek ile eluted. Ayrıca, üretim baculovirus yanı sıra üzerinde yakalama ve elüsyon Kromatografi sütundan yapılabilir mevcut iyi üretim uygulamaları (cGMP) ile uyumlu olan bir kapalı sistem işlemi kullanarak.
Burada, biz üretim, arıtma ve bulaşıcı baculovirus benzeşme Kromatografi ve Santrifüjü kullanarak konsantrasyon için detaylı bir protokol sağlar. Kısaca, baculovirus ile transfection Sf9 hücre yapisan kültür baculovirus plazmid DNA ile üretmek ve daha fazla serum serbest süspansiyon kültürü bulaşıcı baculovirus genişletin. Heparin benzeşme Kromatografi kullanarak baculovirus arındırmak ve ultrasantrifüj vektör gen terapisi uygulama için son derece konsantre için son adım olarak kullanın.

<table border="0" cellpadding="0" cellspacing="0" style="width:784px;" width="784">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="16">
			<td height="16" style="height:16px;width:257px;">Akta Avant 150</td>
			<td style="width:204px;">GE Healthcare</td>
			<td style="width:156px;">28976337</td>
			<td style="width:167px;">Chromatography system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:257px;">POROS Heparin 50 &#181;m Column</td>
			<td>ThermoFisher Scientific</td>
			<td>4333414</td>
			<td>Heparin column</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td>Non-catalog item</td>
			<td>Concentrates virus at high-speed</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Polyallomer Ultracentrifuge tube</td>
			<td>Beckman-Coulter</td>
			<td>326823</td>
			<td>Concentrates virus at high-speed</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MaxQ 8000 orbital shaker incubator&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>Non-catalog item</td>
			<td>Shaker for suspension culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mL Erlenmeyer flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238071</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L Erlenmeyer flasks</td>
			<td>ThermoFisher Scientific</td>
			<td>238072</td>
			<td>Flask for suspension culture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microscope (Olympus CKX41)</td>
			<td>Olympus</td>
			<td>Non-catalog item</td>
			<td>Cell monitoring and counting&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Table top centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75253839/433607</td>
			<td>For clarification of Baculovirus supernatant</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">50 ml Conical tube</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-49A</td>
			<td>For collection of Baculovirus supernatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>07-200-80</td>
			<td>Tissue culture treated plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10-cm plate</td>
			<td>ThermoFisher Scientific</td>
			<td>08-772E</td>
			<td>Tissue culture treated plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Stericup-HV, 0.45&#160;&#181;m, PVDF</td>
			<td>EMD-Millipore</td>
			<td>SCHVU05RE</td>
			<td>Filtration unit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15160-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracycline</td>
			<td>Sigma-Aldrich</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gentamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bac-to-Bac Vector Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>10360-014</td>
			<td>Baculovirus expression system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DH10B-T1R&#160; Competent cell</td>
			<td>ThermoFisher Scientific</td>
			<td>12331-013</td>
			<td>Competent cell for bacmid</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">TE buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>10 mM Tris-HCl, 1 mM EDTA, pH 8.0.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid Maxiprep kit</td>
			<td>ThermoFisher Scientific</td>
			<td>K2100-06</td>
			<td>For bacmid purification</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sf9 Cells</td>
			<td>ThermoFisher Scientific</td>
			<td>11496-015</td>
			<td>Insect cells</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Grace&#8217;s Insect Cell Culture Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11605-094</td>
			<td>Transfection medium</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>20012227</td>
			<td>Washing cells, diluting samples</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:257px;">HyClone SFX-Insect cell media</td>
			<td>GE Healthcare</td>
			<td>SH30278.02</td>
			<td>Serum-free insect cell growth medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Benzonase Nuclease</td>
			<td>Sigma-Aldrich</td>
			<td>E1014</td>
			<td>Enzyme to degrade DNA and RNA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Baculovirus plasmid (bacmid) DNA</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>Bacmid for Baculovirus Production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellfectin II&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>10362</td>
			<td>Transfection reagent for insect cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4737</td>
			<td>Stabilizes Baculovirus</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryovial</td>
			<td>Thomas Scientific</td>
			<td>1222C24</td>
			<td>For storage of Baculovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HT1080 cell line</td>
			<td>ATCC</td>
			<td style="width:156px;">CCL-121</td>
			<td>Fibroblast cell line</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Growth media for cell lines</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Wash buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elution buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Column cleaning buffer</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile water</td>
			<td>In-house</td>
			<td>Non-catalog item</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>1.09137</td>
			<td>For Akta Avant cleaning</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7073</td>
			<td>For Akta Avant cleaning</td>
		</tr>
	</tbody>
</table>

production and purification of baculovirus for gene therapy application

Baculovirus geleneksel olarak rekombinant protein ve aşı üretimi için kullanılmıştır. Ancak, son zamanlarda, baculovirus gen terapisi uygulama için umut verici bir vektör olarak ortaya çıkıyor. Burada, baculovirus bir yapisan Sf9 hücre kültüründe baculovirus plazmid DNA (bacmid) geçici transfection tarafından üretilir. İstenen ses elde edilir kadar Baculovirus daha sonra serum serbest süspansiyon kültür Sf9 hücrelerde genişletilir. Sonra kültür heparin benzeşme Kromatografi kullanarak süpernatant saf. Virüs süpernatant glikoprotein baculovirus örtmek için baculovirus parçacıklar süpernatant heparin yakınlık nedeniyle içinde bağlar heparin sütun yüklenir. Sütun kirletici kaldırmak için bir tampon ile yıkanır ve baculovirus ile bir yüksek-tuz arabellek sütundan eluted. Eluate izotonik bir tuz konsantrasyonu seyreltilmiş ve baculovirus parçacıkları daha fazla ultrasantrifüj kullanarak yoğunlaşmıştır. Bu yöntemi kullanarak, baculovirus bulaşıcı parçacıklar % 25 kurtarma ile ilâ 500-fold konsantre olabilir. Burada açıklanan protokol 1 L kültürlerden üretim ve baculovirus arınma gösterir rağmen yönteminin ölçekli-klinik-grade vektör gen terapisi uygulama için üretmek için bir kapalı sistem süspansiyon kültür up.

Sunulan bir akış diyagramı (şekil 1) protokolüdür. Adımlar Sf9 hücre bacmid baculovirus bir tabak içinde yapisan kültür, serum serbest süspansiyon kültür, nükleaz tedavi ve açıklama Santrifüjü ve filtrasyon tarafından sonraki amplifikasyon üretmek için DNA ile geçici transfection içerir, ve heparin benzeşme Kromatografi kullanarak arıtma konsantrasyon ultrasantrifüj ile izledi.
<p class="jove_content" fo:keep-together.within-pag...

Watch this Scientific Journal Video about Production and Purification of Baculovirus for Gene Therapy Application at JoVE.com

Production and Purification of Baculovirus for Gene Therapy Application

Üretim ve Baculovirus arınma gen tedavisi uygulaması için

Bu protokol için baculovirus baculovirus plazmid geçici transfection Sf9 hücreleri tarafından üretilen ve bir serum serbest süspansiyon kültürde güçlendirilmiş. Süpernatant heparin benzeşme Kromatografi tarafından saf ve ultrasantrifüj tarafından daha da yoğunlaştı. Bu iletişim kuralı üretim ve baculovirus gen terapisi uygulama için arıtma için yararlıdır.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

production-purification-baculovirus-for-gene-therapy

Research

JoVE Journal

Genetics

14.6K Görüntüleme.  Cincinnati Children's Hospital Medical Center. Bu protokol için baculovirus baculovirus plazmid geçici transfection Sf9 hücreleri tarafından üretilen ve bir serum serbest süspansiyon kültürde güçlendirilmiş. Süpernatant heparin benzeşme Kromatografi tarafından saf ve ultrasantrifüj tarafından daha da yoğunlaştı. Bu iletişim kuralı üretim ve baculovirus gen terapisi uygulama için arıtma için yararlıdır.

Video: Production and Purification of Baculovirus for Gene Therapy Application

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Yenilenebilir Biyoyakıt ve Biyokimyasal Üretim için bir Sıradışı Maya Genetik Mühendisliği

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

Genetik

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Integrase-eksik Lentiviral vektörler üretimi için CRISPR/Cas9-aracılı Gene hücre bölünmesi içinde nakavt için bir iletişim kuralı

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Verimli üretim ve Model sistem Danio rerio kimlik CRISPR/Cas9 tarafından oluşturulan Gene altını gizleme

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Fare ve insan hematopoetik progenitör hücre tarafından CRISPR/Cas9 yüksek verimli Gene bozulma

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Hazırlık ve Nonhuman primat hematopoetik kök ve progenitör hücrelerin gen modifikasyonu

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Kardiyak hücre tedavisi için kök hücre DNA bütünlük değerlendirilmesi

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Uzaktan kumandalı sistem in vivo uygulama için işleme endojen gen ekspresyonu

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Dönüşüm, Genom Düzenleme ve Fenotipleme Azot sabitleme Tropikal Cannabaceae Ağaç Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

Glioblastoma'da İşlevsel İlişkili MiRNA'ların Ve Bunların Mühendisliğinin Gen Tedavisi İçin Yapay Kümelere Dönüştürülmesi

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

Genetiği Değiştirilmiş Farelerin Yüksek Verimli Üretimi için Dondurulmuş Çözülmüş Embriyoların Kullanımı

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...