Bir kariyer geliştirme Ödülü gelen Department of Veterans Affairs [BX002797] L.E.W. ve ulusal sağlık Enstitüleri [R01-DK095867] desteklenen ve Amerikan Kalp Derneği [15GRNT25700209] J.C. desteklenen Ulusal Sağlık Enstitüleri [DK093660], Department of Veterans Affairs [BX002190] ve böbrek hastalığı Vanderbilt Merkezi M.H.W. desteklenen Bu malzeme kaynakları ve VA Tennessee Vadisi sağlık sistemine tesislerinin kullanımı ile desteklenen iş sonucudur.

<ol>
	<li>Liu, F., Song, Y., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamics-based+transfection+in+animals+by+systemic+administration+of+plasmid+DNA.">Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.</a> Gene Ther. 6 (7), 1258-1266 (1999).</li><li>Zhang, G., Budker, V., Wolff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+foreign+gene+expression+in+hepatocytes+after+tail+vein+injections+of+naked+plasmid+DNA.">High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.</a> Human Gene Therapy. 10, 1735-1737 (1999).</li><li>Fumoto, S., Nishimura, K., Nishida, K., Kawakami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Imaging+of+the+Intracellular+Fate+of+Plasmid+DNA+and+Transgene+Expression:+ZsGreen1+and+Tissue+Clearing+Method+CUBIC+Are+an+Optimal+Combination+for+Multicolor+Deep+Imaging+in+Murine+Tissues.">Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues.</a> PLoS One. 11 (1), e0148233 (2016).</li><li>Yoshino, H., Hashizume, K., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+plasmid+DNA+transfer+to+the+porcine+liver+using+rapid+injection+with+large+volume.">Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume.</a> Gene Ther. 13 (24), 1696-1702 (2006).</li><li>Kamimura, K., Suda, T., Xu, W., Zhang, G., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided,+lobe-specific+hydrodynamic+gene+delivery+to+swine+liver.">Image-guided, lobe-specific hydrodynamic gene delivery to swine liver.</a> Mol Ther. 17 (3), 491-499 (2009).</li><li>Kamimura, K., Zhang, G., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided,intravascular+hydrodynamic+gene+delivery+to+skeletal+muscle+in+pigs.">Image-guided,intravascular hydrodynamic gene delivery to skeletal muscle in pigs.</a> Mol Ther. 18 (1), 93-100 (2010).</li><li>Suda, T., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+gene+delivery:+its+principles+and+applications.">Hydrodynamic gene delivery: its principles and applications.</a> Mol.Ther. 15 (12), 2063-2069 (2007).</li><li>Alino, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+DNA+delivery+to+whole+pig+cardiac+tissue+by+coronary+sinus+retrograde+injection+employing+non-invasive+catheterization.">Naked DNA delivery to whole pig cardiac tissue by coronary sinus retrograde injection employing non-invasive catheterization.</a> J Gene Med. 12 (11), 920-926 (2010).</li><li>Suda, T., Gao, X., Stolz, D. B., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+impact+of+hydrodynamic+injection+on+mouse+liver.">Structural impact of hydrodynamic injection on mouse liver.</a> Gene Ther. 14 (2), 129-137 (2007).</li><li>Skrypnyk, N. I., Harris, R. C., de Caestecker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+model+of+acute+kidney+injury+and+post+injury+fibrosis+in+mice.">Ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice.</a> J Vis Exp. (78), (2013).</li><li>Woodard, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney-specific+transposon-mediated+gene+transfer+in+vivo.">Kidney-specific transposon-mediated gene transfer in vivo.</a> Sci Rep. 7, 44904 (2017).</li><li>Liang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+glutathione+S-transferase+A4+accelerates+obstruction-induced+tubule+damage+and+renal+fibrosis.">Loss of glutathione S-transferase A4 accelerates obstruction-induced tubule damage and renal fibrosis.</a> Journal of Pathology. 228 (4), 448-458 (2012).</li><li>Liang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+of+insulin-like+growth+factor-1+receptor+in+endothelial+cells+against+unilateral+ureteral+obstruction-induced+renal+fibrosis.">Protective role of insulin-like growth factor-1 receptor in endothelial cells against unilateral ureteral obstruction-induced renal fibrosis.</a> Am J Pathol. 185 (5), 1234-1250 (2015).</li><li>Corridon, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+facilitate+and+monitor+expression+of+exogenous+genes+in+the+rat+kidney+using+plasmid+and+viral+vectors.">A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors.</a> Am J Physiol Renal Physiol. 304 (9), F1217-F1229 (2013).</li><li>Wallace, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+kidney+volume+in+mice+with+polycystic+kidney+disease+by+magnetic+resonance+imaging.">Tracking kidney volume in mice with polycystic kidney disease by magnetic resonance imaging.</a> Kidney Int. 73 (6), 778-781 (2008).</li><li>Woodard, L. E., Wilson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac-ing+models+and+new+therapeutic+strategies.">piggyBac-ing models and new therapeutic strategies.</a> Trends Biotechnol. 33 (9), 525-533 (2015).</li><li>Yeikilis, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamics+based+transfection+in+normal+and+fibrotic+rats.">Hydrodynamics based transfection in normal and fibrotic rats.</a> World J Gastroenterol. 12 (38), 6149-6155 (2006).</li><li>Thalhofer, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+transgenic+Leishmania+parasites+in+a+live+host.">In vivo imaging of transgenic Leishmania parasites in a live host.</a> J Vis Exp. (41), (2010).</li><li>Wooddell, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+damage+after+delivery+of+naked+plasmid+DNA+into+skeletal+muscles+is+batch+dependent.">Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.</a> Hum Gene Ther. 22 (2), 225-235 (2011).</li><li>Crespo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+liver+gene+transfer+mechanism+involves+transient+sinusoidal+blood+stasis+and+massive+hepatocyte+endocytic+vesicles.">Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.</a> Gene Ther. 12 (11), 927-935 (2005).</li><li>Rocca, C. J., Ur, S. N., Harrison, F., Cherqui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rAAV9+combined+with+renal+vein+injection+is+optimal+for+kidney-targeted+gene+delivery:+conclusion+of+a+comparative+study.">rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study.</a> Gene Ther. 21 (6), 618-628 (2014).</li></ol>

Yazarlar ifşa ve hiçbir çıkar çatışması bildirmek üzere bir şey yok.

Bu protokol için tekrarlanabilir gen ekspresyonu özellikle böbrek de ulaşmak için sağlam bir yöntemi açıklanmıştır. Orta derecede deneyimli bir cerrah elinde transgene okuma duyarlılığını ve 50-%100, fare yaş bağlı olarak çeşitli olması için bu tekniği tarafından transfected fareler yüzdesi bulduk. Luciferase gen ekspresyonu düzeyini arka plan üstüne piggyBac Transpozonlar alma farelerde birkaç ay oldu ve tamamen aynı Transpozonlar11alma immün farelerde bi...

Hidrodinamik kuyruk ven enjeksiyon tekniği gen ekspresyonu fare karaciğer1,2yüksek düzeylerine ulaşmak için yaygın olarak kullanılan bir yol haline gelmiştir. Böbrekler de bu tekniği çok daha alt bir düzeyde, yaklaşık 100-fold daha az3ile transfected. Burada açıklanan hidrodinamik renal pelvis enjeksiyon organ ifade daha önce karaciğer4' te,5 kurulmuş aynı hidrodinamik ilkeleri kullanarak fiziksel yollarla özgüllük denetlemek için basit bir yol sağlar , kas6ve diğer organları7,8. Basınç kullanarak canlı hayvan vivo içinde hücrelerde bu yöntem transfects ve aynı anda hızlı bir şekilde organ hasar inducing sıvı içeren DNA hücre zorlamak için hız9çözüldü. Tarafından insülin şırınga ile birlikte tek bir enjeksiyon kanat kesi10 üzerinden böbrek görselleştirmek için köklü cerrahi teknikleri kullanarak, çeşitli böbrek hücre, interstisyel fibroblastlar esas olarak başarılı transfection bulduk, tübüllerin ve toplama kanal11. Bu fareler diseksiyon diğer organ görüntüleme teknikleri11luciferase tarafından görselleştirmek için yüksek düzeyde transfected değil göstermiştir. Teknik olmayan viral olduğu için plazmid DNA kullanımına transfection için enjeksiyon için gerekli reaktifler hızlı ve kolay hazırlanması izin verir.
Antioksidan glutatyon S-transferaz A4, insülin benzeri büyüme faktörü-1 reseptör ve böbrek beklenen biyolojik etkileri11, ile tüm hormon Eritropoetin ifade için yerelleştirilmiş hidrodinamik iğneleri kullandık 12 , 13. yönetim, enjeksiyon hacmi, DNA dozaj ve organizatörü seçim rotanın ayrıntılı değerlendirme gerçekleştirilen11oldu. Ayrıca, bağışıklık reaksiyonu transgene için bastırmak için her iki piggyBac transposon sistemi ve/veya siklofosfamid tedavisi gösterilmiştir uzun vadeli gen ifadesi sonuçlar11geliştirmek için. Diğer araştırmacılar renal ven yaklaşım saat bir ay14daha büyük bir süre için yüksek transfection verimliliği elde başarı, fareyle kullanmışlardır. Ancak, genetik insan hastalığı taklit fenotipleri düzeltilmesi genellikle yapılma farelerde ilk kanıtı-of-concept olarak en memeli genetik modellerini fare modelleri olduğundan. Renal ven enjeksiyon renal pelvis enjeksiyon için karşılaştırıldığında ve renal pelvis içine enjeksiyon renal ven gen ekspresyonu (yaklaşık on daha yüksek) ve hayatta kalma11için daha üstün olduğunu ortaya koymuştur. Renal pelvis idrar üretim dalgalanmalar tolere esnek olduğu için böbrek girdiği ideal bir yoldur ve sık sık ne zaman bile hydronephrosis sırasında genişlemiş, yapısal bütünlüğünü korumak yapabiliyor. Ayrıca, renal pelvis içine enjeksiyon böbrek böbrek kapsülü enjekte sıvı gözle görülür tarafından böbrek intraparenchymal enjeksiyon iyi korunabilmesi izin, piercing olmadan erişebileceğini. Diğer fare organlara girişinin damarlara dışında bir yol var mı ama bir ideal enjeksiyon izi böbrek idrar alanıdır. Ayrıca, renal ven içine enjeksiyon karın boşluğuna kan sızıntı sonuçlandı. Vahşi tipi fare böbrekler toplam böbrek hacmi yaklaşık 0.2 cm3, tek böbrek hacmi yaklaşık renal pelvis tarafından hidrodinamik enjekte sıvı miktarı eşittir olmak manyetik rezonans görüntüleme tarafından tahmin edilmektedir enjeksiyon (100 µL)15. Burada, tüm böbrek tekrarlanabilir transfection elde etmek için hidrodinamik renal pelvis enjeksiyon protokolünün detaylı nüanslar mevcut yaptık.

<table>
	<tbody>
		<tr>
			<td>AnaSed Xylazine</td>
			<td>Patterson Veterinary</td>
			<td>07-808-1947</td>
			<td>Anesthetic - Not controlled substance</td>
		</tr>
		<tr>
			<td>BD Insulin Syringe 0.5 mL 29G 1/2 Inch</td>
			<td>Cardinal Health</td>
			<td>309306</td>
			<td>Required syringes</td>
		</tr>
		<tr>
			<td>Buprenex</td>
			<td>Pharmacist/Veterinarian</td>
			<td></td>
			<td>Analgesia - Controlled Substance</td>
		</tr>
		<tr>
			<td>Dynarex Disposable Towel Drape</td>
			<td>Thermo Fisher Scientific</td>
			<td>19-310-671</td>
			<td>Place over heat pad</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>Use only endotoxin-free plasmid DNA</td>
		</tr>
		<tr>
			<td>Endosafe Gel-Clot LAL Rapid Positive Control</td>
			<td>Charles River</td>
			<td>PC200</td>
			<td>Positive control for endotoxin test</td>
		</tr>
		<tr>
			<td>Endosafe Gel-Clot LAL Rapid Single Test Vial</td>
			<td>Charles River</td>
			<td>R13500</td>
			<td>Endotoxin test</td>
		</tr>
		<tr>
			<td>Extra Fine Micro Dissecting Scissors</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5882</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Fisherbrand Instant Sealing Sterilization Pouch - 9&#34;</td>
			<td>Thermo Fisher Scientific</td>
			<td>01-812-51</td>
			<td>For autoclaving surgical tools</td>
		</tr>
		<tr>
			<td>Gaymar Heat Pump</td>
			<td>Paragon Medical</td>
			<td>TP-700</td>
			<td>Water-circulating heat pump</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>Roboz Surgical Instrument</td>
			<td>DS-401</td>
			<td>To reuse surgical tools during surgery</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5136</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Graefe Tissue Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5153</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Halsey Needle Holder, 5&#34; Length</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-7841</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Heat pads - 15&#34; x 21&#34; - need at least 3</td>
			<td>Paragon Medical</td>
			<td>TP22G</td>
			<td>For use with Gaymar Heat Pump</td>
		</tr>
		<tr>
			<td>IsoFlo (Isoflurane, USP)</td>
			<td>Abbott Animal Health</td>
			<td>5260-04-05</td>
			<td>For imaging and euthanasia</td>
		</tr>
		<tr>
			<td>Isotec Isoflurane Delivery System Vaporizor</td>
			<td>Smiths Medical</td>
			<td>VCT3K2</td>
			<td>For imaging and euthanasia</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Pharmacist/Veterinarian</td>
			<td></td>
			<td>Anesthetic - Controlled Substance</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34120</td>
			<td>Laboratory tissues</td>
		</tr>
		<tr>
			<td>Living Image software</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td>For live animal imaging</td>
		</tr>
		<tr>
			<td>Luciferin</td>
			<td>Perkin Elmer</td>
			<td>122796</td>
			<td>For live animal imaging</td>
		</tr>
		<tr>
			<td>Nanodrop 2000</td>
			<td>Thermo Scientific</td>
			<td>ND-2000-US-CAN</td>
			<td>Spectrophotometer for DNA measurement</td>
		</tr>
		<tr>
			<td>Prevantics Swabs</td>
			<td>Thermo Fisher Scientific</td>
			<td>23-100-110</td>
			<td>For skin surgery prep</td>
		</tr>
		<tr>
			<td>Prolene 5-0 sutures Taper 30&#34;</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC0256891</td>
			<td>Non-absorbable sutures for skin</td>
		</tr>
		<tr>
			<td>Puralube Brand Opthalmic Ointment</td>
			<td>Patterson Veterinary</td>
			<td>07-888-2572</td>
			<td>To keep eyes moist during surgery</td>
		</tr>
		<tr>
			<td>Trans IT - QR Hydrodynamic Delivery Solution</td>
			<td>Mirus Bio</td>
			<td>MIR-5240</td>
			<td>Hydrodynamic delivery buffer for diluting DNA</td>
		</tr>
		<tr>
			<td>Vicryl 5-0 Sutures J303H</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9816710</td>
			<td>Absorbable sutures for muscle layer</td>
		</tr>
		<tr>
			<td>Wahl Mini Arco Clipper</td>
			<td>Med-Vet International</td>
			<td>8787-1550</td>
			<td>Shaver for skin prep</td>
		</tr>
		<tr>
			<td>Xenogen IVIS 200</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td>For live animal imaging</td>
		</tr>
	</tbody>
</table>

hydrodynamic renal pelvis injection for non-viral expression of proteins in the kidney

Hidrodinamik enjeksiyon plazmid DNA ve diğer maddeler ile çeşitli dokularda transfect için yerel, yüksek basınçlı bir ortam oluşturur. Hidrodinamik kuyruk ven enjeksiyon, örneğin, hangi karaciğer transfected iyi kurulmuş bir yöntemdir. Bu el yazması plazmid DNA için böbrek özel gen ekspresyonu ile doğrudan fare böbrek iğne ile bir uygulama hidrodinamik ilkeleri açıklar. Fareler anestezi ve böbrek renal pelvis doğrudan bir plazmid DNA içeren çözüm hızlı bir iğne ile takip bir kanat kesi tarafından yararlanılır. İğneyi yerinde on saniye tutulur ve kesi sitesi dikmiş. Ertesi gün, hayvan görüntüleme live, Western blot veya immünhistokimya gen ekspresyonu tahlil için kullanılabilir veya diğer deneyleri seçim transgene için uygun faiz protein tespiti için kullanılır. Gen ifadesinin uzatmak için yayımlanmış yöntemleri transgene için bağışıklık yanıtını inhibe transposon-aracılı transgene entegrasyon ve siklofosfamid tedavisi içerir.

Cerrahi ve enjeksiyon tekniği gerçekleştirmek basit bir kez hakim, hiçbir büyük ekipman veya pahalı malzemeler gerektiren. Yeni Eğer kanat-kesi böbrek ameliyatı, birkaç fare ötenazi için zamanlanmış bir eğitim gün bu ameliyatı ilk denemesinde normalden daha uzun sürebilir çünkü fareler kurtarılan aşağıdaki ameliyat olan değil izin verilmelidir. Alternatif olarak, müfettişler benzer teknikleri ile tanıdık daha çok basit gelebilir. Şekil...

Watch this Scientific Journal Video about Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney at JoVE.com

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

Hidrodinamik Renal Pelvis enjeksiyon böbrek proteinlerin Non-viral ifade

Bu iletişim kuralı yoluyla renal pelvis transgene özellikle böbrek ifadede üretmek için fare böbrek içine plazmid DNA enjekte yöntemi açıklanır.

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...

hydrodynamic-renal-pelvis-injection-for-non-viral-expression-proteins

Research

JoVE Journal

Biology

18.3K Görüntüleme.  Tennessee Valley Healthcare System. Bu iletişim kuralı yoluyla renal pelvis transgene özellikle böbrek ifadede üretmek için fare böbrek içine plazmid DNA enjekte yöntemi açıklanır.

Video: Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

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

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

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

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

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

Biyoloji

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murin Böbrek Nakli Prosedürü

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Rekombinant Proteinlerin Metilotropik maya İfade Pichia pastoris</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

In vitro Reconstitution of the Active T. castaneum Telomerase

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

In vitro Reconstitution of the Active T. castaneum Telomerase

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

Active T. in vitro Sulandırma castaneum Telomeraz

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

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Rekombinant Proteinlerin Yüksek-düzey Geçici İfade için Bitkiler Verimli Agroinfiltration

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Farelerde Hidrodinamik Gen Taşıyıcı Proteinlerin Geçici İfadesi

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

İfadesi için HeLa Tabanlı Hücre Özgür İfade Sistemleri<em&gt; Plasmodium</em&gt; Rhoptry Proteinler

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

Süspansiyon HEK293 hücrelerinin kısa süreli transfection ile Rekombinant İnsan Proteinlerinin Yüksek Verimli İfadesi

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

İzole Çalışma Sıçan Kalp Glukoz ve Yağ Asidi Oksidasyonu Oranlarının Belirlenmesine Yöntemleri

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...