Name: transient expression of proteins by hydrodynamic gene delivery in mice
Uploaded: 2014-05-05T16:30:00
Description: Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

نشكر ليو شيا (Regeneron صيدلة) ليعلمنا هذا الأسلوب في البداية HGD والدكتور راسيل طومسون (كلية ألبرت اينشتاين للطب) للتوجيه المستمر له في مختلف جوانب التكنولوجيا. وقد تم تمويل هذا العمل من قبل كلية هانتر، جامعة مدينة نيويورك بدء الأموال والجائزة NSF الخبز IOS-1249166. نشكر NYULMC التشريح المرضي مركز كور NYUCI دعم غرانت، المعاهد الوطنية للصحة / NCI 5 P30CA16087-31 لالأنسجة في كبد الفأر.

تمت الموافقة على جميع التجارب وصفها هنا من قبل اللجنة المؤسسية رعاية الحيوان واستخدام كلية هانتر، جامعة مدينة نيويورك. 1. إعداد الحمض النووي البلازميد خالية من الذيفان الداخلي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اخ...

<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, 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> Hum Gene Ther. 10, 1735-1737 (1999).</li><li>Miao, C. H., Thompson, A. R., Loeb, K., Ye, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+and+therapeutic-level+hepatic+gene+expression+of+human+factor+IX+after+naked+plasmid+transfer+in+vivo.">Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo.</a> Mol Ther. 3, 947-957 (2001).</li><li>Magin-Lachmann, C., 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+vitro+and+in+vivo+delivery+of+intact+BAC+DNA+--+comparison+of+different+methods.">In vitro and in vivo delivery of intact BAC DNA -- comparison of different methods.</a> J Gene Med. 6, 195-209 (2004).</li><li>Chang, J., Sigal, L. J., Lerro, A., Taylor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+the+human+hepatitis+delta+virus+genome+Is+initiated+in+mouse+hepatocytes+following+intravenous+injection+of+naked+DNA+or+RNA+sequences.">Replication of the human hepatitis delta virus genome Is initiated in mouse hepatocytes following intravenous injection of naked DNA or RNA sequences.</a> J Virol. 75, 3469-3473 (2001).</li><li>Giladi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+interfering+RNA+inhibits+hepatitis+B+virus+replication+in+mice.">Small interfering RNA inhibits hepatitis B virus replication in mice.</a> Mol Ther. 8, 769-776 (2003).</li><li>Kobayashi, N., 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=Vector-based+in+vivo+RNA+interference:+dose-+and+time-dependent+suppression+of+transgene+expression.">Vector-based in vivo RNA interference: dose- and time-dependent suppression of transgene expression.</a> J Pharmacol Exp Ther. 308, 688-693 (2004).</li><li>Layzer, J. 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=In+vivo+activity+of+nuclease-resistant+siRNAs.">In vivo activity of nuclease-resistant siRNAs.</a> Rna. 10, 766-771 (2004).</li><li>McCaffrey, A. 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=RNA+interference+in+adult+mice.">RNA interference in adult mice.</a> Nature. 418, 38-39 (2002).</li><li>McCaffrey, A. 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=Determinants+of+hepatitis+C+translational+initiation+in+vitro,+in+cultured+cells+and+mice.">Determinants of hepatitis C translational initiation in vitro, in cultured cells and mice.</a> Mol Ther. 5, 676-684 (2002).</li><li>McCaffrey, A. P., Meuse, L., Karimi, M., Contag, C. H., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potent+and+specific+morpholino+antisense+inhibitor+of+hepatitis+C+translation+in+mice.">A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice.</a> Hepatology. 38, 503-508 (2003).</li><li>Zhang, G., 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=Hydroporation+as+the+mechanism+of+hydrodynamic+delivery.">Hydroporation as the mechanism of hydrodynamic delivery.</a> Gene Ther. 11, 675-682 (2004).</li><li>Kobayashi, N., Kuramoto, T., Yamaoka, K., Hashida, M., Takakura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+uptake+and+gene+expression+mechanisms+following+intravenous+administration+of+plasmid+DNA+by+conventional+and+hydrodynamics-based+procedures.">Hepatic uptake and gene expression mechanisms following intravenous administration of plasmid DNA by conventional and hydrodynamics-based procedures.</a> J Pharmacol Exp Ther. 297, 853-860 (2001).</li><li>Kobayashi, N., Nishikawa, M., Hirata, K., Takakura, Y. <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+procedure+involves+transient+hyperpermeability+in+the+hepatic+cellular+membrane:+implication+of+a+nonspecific+process+in+efficient+intracellular+gene+delivery.">Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery.</a> J Gene Med. 6, 584-592 (2004).</li><li>Bonamassa, B., Hai, L., 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+and+its+applications+in+pharmaceutical+research.">Hydrodynamic gene delivery and its applications in pharmaceutical research.</a> Pharm Res. 28, 694-701 (2011).</li><li>Al-Dosari, M. S., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+gene+delivery:+principle,+limitations,+and+recent+progress.">Nonviral gene delivery: principle, limitations, and recent progress.</a> Aaps J. 11, 671-681 (2009).</li><li>Gao, X., Kim, K. S., 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=Nonviral+gene+delivery:+what+we+know+and+what+is+next.">Nonviral gene delivery: what we know and what is next.</a> Aaps J. 9, (2007).</li><li>Herweijer, H., 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=Gene+therapy+progress+and+prospects:+hydrodynamic+gene+delivery.">Gene therapy progress and prospects: hydrodynamic gene delivery.</a> Gene Ther. 14, 99-107 (2007).</li><li>Kobayashi, N., Nishikawa, M., Takakura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydrodynamics-based+procedure+for+controlling+the+pharmacokinetics+of+gene+medicines+at+whole+body,+organ+and+cellular+levels.">The hydrodynamics-based procedure for controlling the pharmacokinetics of gene medicines at whole body, organ and cellular levels.</a> Adv Drug Deliv Rev. 57, 713-731 (2005).</li><li>Al-Dosari, M. S., Knapp, J. E., 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+delivery.">Hydrodynamic delivery.</a> Adv Genet. 54, 65-82 (2005).</li><li>Bell, J. B., 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=Preferential+delivery+of+the+Sleeping+Beauty+transposon+system+to+livers+of+mice+by+hydrodynamic+injection.">Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection.</a> Nat Protoc. 2, 3153-3165 (2007).</li><li>Osorio, F. G., de la Rosa, J., Freije, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminescence-based+in+vivo+monitoring+of+NF-kappaB+activity+through+a+gene+delivery+approach.">Luminescence-based in vivo monitoring of NF-kappaB activity through a gene delivery approach.</a> Cell Commun Signal. 11, (2013).</li><li>Knapp, J. E., 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+delivery+of+DNA.">Hydrodynamic delivery of DNA.</a> Methods Mol Biol. 245, 245-250 (2004).</li><li>Yang, J., Chen, S., Huang, L., Michalopoulos, G. K., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+expression+of+naked+plasmid+DNA+encoding+hepatocyte+growth+factor+in+mice+promotes+liver+and+overall+body+growth.">Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.</a> Hepatology. 33, 848-859 (2001).</li><li>Maruyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+expression+of+naked+DNA+delivered+to+rat+liver+via+tail+vein+injection.">High-level expression of naked DNA delivered to rat liver via tail vein injection.</a> J Gene Med. 4, 333-341 (2002).</li><li>Hen, G., 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=Expression+of+foreign+genes+in+chicks+by+hydrodynamics-based+naked+plasmid+transfer+in+vivo.">Expression of foreign genes in chicks by hydrodynamics-based naked plasmid transfer in vivo.</a> Domestic animal endocrinology. 30, 135-143 (2006).</li><li>Eastman, S. 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=Development+of+catheter-based+procedures+for+transducing+the+isolated+rabbit+liver+with+plasmid+DNA.">Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA.</a> Hum Gene Ther. 13, 2065-2077 (2002).</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, 1696-1702 (2006).</li><li>Vanden Bossche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+general+aspects+of+the+distribution+and+epidemiology+of+bovine+trypanosomosis+in+southern+Africa.">Some general aspects of the distribution and epidemiology of bovine trypanosomosis in southern Africa.</a> Int J Parasitol. 31, 592-598 (2001).</li><li>Fevre, E. M., Picozzi, K., Jannin, J., Welburn, S. C., Maudlin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis:+Epidemiology+and+control.">Human African trypanosomiasis: Epidemiology and control.</a> Adv Parasitol. 61, 167-221 (2006).</li><li>Lugli, E. B., Pouliot, M., Portela Mdel, P., Loomis, M. R., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primate+trypanosome+lytic+factors.">Characterization of primate trypanosome lytic factors.</a> Mol Biochem Parasitol. 138, 9-20 (2004).</li><li>Pays, 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=The+trypanolytic+factor+of+human+serum.">The trypanolytic factor of human serum.</a> Nat Rev Microbiol. 4, 477-486 (2006).</li><li>Raper, J., Fung, R., Ghiso, J., Nussenzweig, V., Tomlinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+trypanosome+lytic+factor+from+human+serum.">Characterization of a novel trypanosome lytic factor from human serum.</a> Infect Immun. 67, 1910-1916 (1999).</li><li>Vanhamme, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+L-I+is+the+trypanosome+lytic+factor+of+human+serum.">Apolipoprotein L-I is the trypanosome lytic factor of human serum.</a> Nature. 422, 83-87 (2003).</li><li>Nielsen, M. 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=Haptoglobin-related+protein+is+a+high-affinity+hemoglobin-binding+plasma+protein.">Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein.</a> Blood. 108, 2846-2849 (2006).</li><li>Molina-Portela Mdel, P., Lugli, P., Recio-Pinto, E., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosome+lytic+factor,+a+subclass+of+high-density+lipoprotein,+forms+cation-selective+pores+in+membranes.">Trypanosome lytic factor, a subclass of high-density lipoprotein, forms cation-selective pores in membranes.</a> Mol Biochem Parasitol. 144, 218-226 (2005).</li><li>Perez-Morga, D., 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=Apolipoprotein+L-I+promotes+trypanosome+lysis+by+forming+pores+in+lysosomal+membranes.">Apolipoprotein L-I promotes trypanosome lysis by forming pores in lysosomal membranes.</a> Science. 309, 469-472 (2005).</li><li>Molina-Portela, M. P., Samanovic, M., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+of+apolipoprotein+components+within+the+trypanosome+lytic+factor+complex+revealed+in+a+novel+transgenic+mouse+model.">Distinct roles of apolipoprotein components within the trypanosome lytic factor complex revealed in a novel transgenic mouse model.</a> J Exp Med. 205, 1721-1728 (2008).</li><li>Oli, M. W., Cotlin, L. F., Shiflett, A. M., Hajduk, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+resistance-associated+protein+blocks+lysosomal+targeting+of+trypanosome+lytic+factor+in+Trypanosoma+brucei.">Serum resistance-associated protein blocks lysosomal targeting of trypanosome lytic factor in Trypanosoma brucei.</a> Eukaryot Cell. 5, 132-139 (2006).</li><li>Thomson, R., Molina-Portela, P., Mott, H., Carrington, M., Raper, J. <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+of+baboon+trypanosome+lytic+factor+eliminates+both+animal+and+human-infective+African+trypanosomes.">Hydrodynamic gene delivery of baboon trypanosome lytic factor eliminates both animal and human-infective African trypanosomes.</a> Proc Natl Acad Sci U S A. 106, 19509-19514 (2009).</li><li>Genovese, G., 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=Association+of+trypanolytic+ApoL1+variants+with+kidney+disease+in+African+Americans.">Association of trypanolytic ApoL1 variants with kidney disease in African Americans.</a> Science. 329, 841-845 (2010).</li><li>Tzur, S., 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=Missense+mutations+in+the+APOL1+gene+are+highly+associated+with+end+stage+kidney+disease+risk+previously+attributed+to+the+MYH9+gene.">Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene.</a> Hum Genet. 128, 345-350 (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, 129-137 (2007).</li></ol>

عندما يقوم بشكل صحيح، HGD هو وسيلة آمنة وفعالة بشكل ملحوظ التسليم التحوير. الخطوات الحاسمة لنجاح HGD هي: 1) تسليم كمية مناسبة من الحمض النووي في كمية كبيرة من المركبات المالحة 2) في الوريد ذيل الفأر 3) في أقل من 8 ثوان. على الرغم من أن عمل...

ليو وآخرون. وتشانغ وآخرون، أصبح إيصال الجينات الهيدروديناميكية (HGD) منذ أول وصف من قبل أداة لا تقدر بثمن لدراسة وظائف الجينات في نظم نموذج القوارض 1، 2. والأسلوب الذي ينطوي على حقن السريع (5-7 ثانية) من حجم كبير (8-12٪ من وزن الجسم) من الحل في الوريد ذيل الفئران لتسهيل امتصاص الحمض النووي البلازميد بواسطة خلايا الأعضاء المستهدفة 1، 2. هذه الظروف تؤدي إلى التعبير الجيني قوية في الكبد وأقل التعبير الجيني في الكلى والطحال والرئة والقلب. حقن 10 ميكروغرام pCMV-LacZ البلازميد يمكن بالنقل بقدر 40٪ من خلايا الكبد، مما يجعل HGD الأكثر كفاءة، غير الفيروسية، في الجسم الحي طريقة إيصال الجينات إلى تاريخ 1. على عكس شركات الطيران الفيروسية، PDNA هي سهلة التحضير، لا تثير استجابة مناعية في المضيف القوارض 3 و لا تشكل مخاطر على الصحة من خلال إعادة مزج مع نهايةogenous الفيروسات. بالإضافة إلى ذلك، منذ جزيئات الحمض النووي التي ألقاها HGD لا تحتاج التعبئة والتغليف، وهذا الأسلوب هو مناسبة لإيصال الكروموسومات البكتيرية المصطنعة (BAC) كبيرة مثل 150 كيلو بايت 4. أنواع أخرى من الجزيئات التي تم تسليمها من خلال طريقة الهيدروديناميكية تشمل الحمض النووي الريبي 5-10، morpholinos 11، والبروتينات 12 و 13 و الجزيئات الصغيرة الأخرى 12 و 14. وقد نوقشت مزايا ومساوئ HGD أكثر من غيرها من طرق التسليم في الاستعراضات ممتازة في الأدب 15-20 وقدمت عددا من الكتاب وصفا تفصيليا للإجراءات 21-23. إدخال الجينات المحورة في الفئران عن طريق HGD هي آمنة وفعالة 1-3، 24، واستخدمت الأسلوب في الفئران مقارنة مع النجاح 25. مع بعض التعديلات، وقد أجريت إثبات صحة مفهوم التجارب في الدجاج 26، راببت 27 و 28 الخنازير، على الرغم من أن في الجسم الحي تطبيق هذه التقنية في الحيوانات الكبيرة لا تزال تشكل تحديا. عند استخدام هذا الأسلوب، الحد آخر شائع هو أن العديد من ناقلات التعبير الثدييات المتاحة تفتقر إلى المكونات لتحقيق الثابتة، ومستوى عال من التعبير الجيني. باستخدام pCMV لوك البلازميد، التعبير الجيني في الأجهزة المستهدفة هو واضح في وقت مبكر بعد عشر دقائق HGD، ومع ذلك، يسقط الأولية، وارتفاع مستوى التعبير بشكل حاد في الأسبوع الأول بعد الحقن 1. طويلة الأجل التعبير التحوير من الممكن اعتمادا على المروج وإنترون المستخدمة في تصميم البلازميد 3، 24 الحقن ومع ذلك، والحفاظ على التعبير الجيني على مستوى عال في كثير من الأحيان يتطلب المتكررة. لهذا السبب، HGD قد تكون أقل مناسبة لدراسة الأمراض المزمنة التي هي نتيجة التعرض الطويل الأمد للبروتينات ضارة أو منتجات البروتين. مع هذه القيود، HGD هو أداة قوية بشكل استثنائي لدراسة بودور tential من الجينات وتأثير ذلك المسوخ في الجسم الحي، فضلا عن الآثار العلاجية وتنظيم البروتينات وإنشاء نماذج حيوانية من المرض (للمراجعة، انظر 15). على سبيل المثال، HGD يمكن أن تستخدم لتعيين وظيفة إلى مجالات والأحماض الأمينية للبروتينات بشكل فردي عن طريق إدخال مختلف بنيات الجينات في الفئران التي طرقت الجينات المعنية بها. علاوة على ذلك، هذه التقنية يمكن استخدامها في أي سلالة الماوس. يصف هذا البروتوكول HGD في الفئران مع التركيز على الجوانب الفنية اللازمة لتحقيق النجاح ترنسفكأيشن: غرز الإبرة الصحيح في الوريد، الحقن حجم وسرعة التسليم. ويتجلى في تطبيق هذا الأسلوب في نموذج الفأر من داء المثقبيات الأفريقي، وهو مرض قاتل للبشر والماشية 29، 30. بينما عدة أنواع من المثقبيات يسبب مرض في الماشية، ومعظم لا يمكن أن تسبب المرض لدى البشر بسبب المركبات المناعية الفطرية في بlood تسمى عوامل التحللي المثقبيات (TLFs) 29، 31، 32. تشكيل هذه المسام، والبروتينات الدهنية عالية الكثافة (HDL) تحتوي على اثنين فريدة من نوعها، الرئيسيات محددة البروتينات: HPR، يجند، مما يسهل امتصاص TLFs في المثقبيات، وAPOL-I، المكون التحللي 31، 33-38 المثقبية البروسية الروديسية قادر على إصابة البشر بسبب التعبير عن المقاومة المرتبطة البروتين مصل (SRA) التي تربط لويحيد الإنسان APOL-I 34، 39. لم يتم تحييد البابون TLF بواسطة حساب الاحتياطي الخاص نظرا لاختلاف APOL-I البروتين 40. كما ذكرت سابقا، وذلك باستخدام ناقلات التعبير الثدييات (pRG977)، والتعبير المعدلة وراثيا من البابون مكونات TLF في الفئران تمنح حماية ضد المثقبيات للعدوى الإنسان 40. إظهار بيانات تمثيلية المقدمة هنا كيف يمكن تطبيق إيصال الجينات الهيدروديناميكية لدراسة الآثار العلاجية للبروتين.

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alcohol Prep Wipe</td>
			<td style="width:113px;">Webcol</td>
			<td align="right" style="width:119px;">6818</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AST kit, Amplite Colorimetric</td>
			<td style="width:113px;">AAT Bioquest</td>
			<td align="right" style="width:119px;">13801</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical Tube, 50 ml</td>
			<td style="width:113px;">BD Falcon</td>
			<td align="right" style="width:119px;">352070</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:113px;">Qiagen</td>
			<td align="right" style="width:119px;">12362</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KimWipes</td>
			<td style="width:113px;">KIMTECH</td>
			<td align="right" style="width:119px;">34120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Mouse Tail Illuminator</td>
			<td style="width:113px;">Braintree Scientific</td>
			<td style="width:119px;">MTI RST</td>
			<td style="width:167px;">Or equivalent</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 20 Gx 1 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305175</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 27 Gx 1/2 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305109</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">pRG977 (mammalian expression vector)</td>
			<td style="width:113px;">Regeneron Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Material Transfer Agreement required</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium Chloride, 0.9% INJ USP 10 ml, Hospira&#160;</td>
			<td style="width:113px;">Fisher</td>
			<td style="width:119px;">NC9054335</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Syringe, 3ml, Luer-Lok Tip</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">309657</td>
			<td style="width:167px;">Luer Lock necessary to withstand pressure from HGD</td>
		</tr>
	</tbody>
</table>

transient expression of proteins by hydrodynamic gene delivery in mice

التعبير كفاءة من الجينات المحورة في الجسم الحي هو من الأهمية بمكان في دراسة وظائف الجينات وتطوير علاجات للأمراض. على مدى السنوات الماضية، والتسليم الجين الهيدروديناميكية (HGD) برز بوصفه وسيلة بسيطة وسريعة وآمنة وفعالة لإيصال الجينات المحورة في القوارض. تعتمد هذه التقنية على القوة الناتجة عن حقن السريع لكمية كبيرة من محلول الفسيولوجية لزيادة نفاذية أغشية الخلايا من الأجهزة perfused وبالتالي تقديم الحمض النووي في الخلايا. واحدة من المزايا الرئيسية لHGD هو القدرة على إدخال الجينات المحورة في خلايا الثدييات باستخدام الحمض النووي البلازميد عاريا (PDNA). إدخال جين الخارجية باستخدام البلازميد هو شاقة الحد الأدنى، كفاءة عالية و، خلافا لناقلات الفيروسية وآمنة بشكل ملحوظ. واستخدمت في البداية لتقديم HGD الجينات في الفئران، ويتم استخدامه الآن لتقديم مجموعة واسعة من المواد، بما في ذلك [أليغنوكليوتيد، الكروموسومات الاصطناعية، RNA، البروتينات والجزيئات الصغيرة في الفئران والجرذانو، إلى درجة محدودة، والحيوانات الأخرى. يصف هذا البروتوكول HGD في الفئران، ويركز على ثلاثة جوانب رئيسية للطريقة التي تعتبر بالغة الأهمية لتنفيذ الإجراء بنجاح: الإدراج الصحيح للإبرة في الوريد، الحقن حجم وسرعة التسليم. يتم إعطاء أمثلة لإظهار تطبيق هذا الأسلوب إلى التعبير عابرة من اثنين من الجينات التي تكود يفرز والبروتينات الرئيسيات محددة، ئي LI (APOL-I) والبروتين المتصلة هابتوغلوبين (HPR).

الموضع الصحيح من الإبرة في الوريد ذيل الماوس (كما هو موضح في الشكل رقم 1) هو شرط أساسي لتقديم بنجاح التحوير من قبل ترنسفكأيشن مقرها الهيدروناميكا. في كثير من الأحيان الجزء الأكثر تحديا للتقنية، ومع ذلك، هو الإبقاء على إبرة داخل الوريد الذيل دون حركة بحيث يمكن...

Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

في ترنسفكأيشن المجراة من الحمض النووي عارية عن طريق التسليم الجين الهيدروديناميكية يدخل الجينات في الأنسجة حيوان مع الحد الأدنى من الاستجابة الالتهابية. تتولد كميات كافية من الجين المنتج بحيث ظيفة الجين والتنظيم فضلا عن بنية البروتين وظيفة يمكن تحليلها.

التعبير عابرة من البروتينات عن طريق هيدرودينامي الجينات في الفئران التسليم

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

transient-expression-of-proteins-by-hydrodynamic-gene-delivery-in-mice

Research

JoVE Journal

Biology

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.

التحضير لزراعة الخلايا المكونة للدم الابتدائية من نخاع عظام الفئران لElectroporation

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

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.

تلطيخ من البروتينات في الهلام مع Coomassie G - 250 دون عضوي حمض الخليك والمذيبات

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

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

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 (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

باستخدام الجينات Electroporation MXcell بولسير النظام لTransfect الخلايا الأولية ذات الكفاءة العالية

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

باستخدام خلية مكافحة الآلي لتبسيط دراسات التعبير الجيني : ضربة قاضية سيرنا من IL - 4 التعبير الجيني في الخلايا التابعة Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

البارافين ، جزءا لا يتجزأ من والمجمدة أقسام ذبابة الفاكهة</emالعضلات الكبار>

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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.

إعادة إعماره في المختبر من أحدث T. castaneum تيلوميراز

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.

Agroinfiltration كفاءة من النباتات للتعبير عابر الرفيع المستوى من البروتينات المؤتلف

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

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

تحليل الحمض النووي الريبي العالمي التجميعي على مستوى خلية واحدة نقص الأكسجة التالي

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

قياس معدل الأيض في<em&gt; ذبابة الفاكهة</em&gt; باستخدام قياس التنفس

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

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.