Name: in vivo nanovector delivery of a heart-specific microrna-sponge
Uploaded: 2018-06-15T15:00:00
Description: Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

Мы благодарим Энтони K. L. Leung Кафедра биохимии и молекулярной биологии, Блумбергской школой общественного здравоохранения, Университет Джона Хопкинса для его технической помощи с проектирование конструкции мир-181-Губка. Мы также благодарим Полина Сыса-Шах и Кэтлин Габриелсон отдела молекулярной и сравнительной Pathobiology, Джонс Хопкинс медицинских учреждений для их технической помощи изображений в естественных условиях доставки Мирна Губка.
Эта работа было поддержано грантов из низ, HL39752 (для Charles Steenbergen) и ученый развития гранта от Американской ассоциации сердца 14SDG18890049 (до Samarjit дас). Крыса кардио конкретной промоутера щедро предоставляемые Джеффри D. Molkentin в Цинциннати детской больнице.

Все экспериментальные процедуры были утверждены институциональный уход животных и использование комитета университета Джона Хопкинса.
1. Губка дизайн
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МикроРНК, привязка 3' УТР Примечание: A Мирна функций посредством конкретных базовых спаривания взаимо...

<ol>
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C., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-181+and+metabolic+regulation+in+the+immune+system.">miR-181 and metabolic regulation in the immune system.</a> Cold Spring Harbor Symposia on Quantitative Biology. 78, 223-230 (2013).</li><li>Hori, 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=miR-181b+regulates+vascular+stiffness+age+dependently+in+part+by+regulating+TGF-beta+signaling.">miR-181b regulates vascular stiffness age dependently in part by regulating TGF-beta signaling.</a> PLoS One. 12 (3), e0174108 (2017).</li><li>Ebert, M. S., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+sponges:+progress+and+possibilities.">MicroRNA sponges: progress and possibilities.</a> RNA. 16 (11), 2043-2050 (2010).</li><li>Ma, 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=Therapeutic+silencing+of+miR-10b+inhibits+metastasis+in+a+mouse+mammary+tumor+model.">Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model.</a> Nature Biotechnology. 28 (4), 341-347 (2010).</li><li>Davis, S., Lollo, B., Freier, S., Esau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+targeting+of+miRNA+with+antisense+oligonucleotides.">Improved targeting of miRNA with antisense oligonucleotides.</a> Nucleic Acids Research. 34 (8), 2294-2304 (2006).</li><li>Davis, 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=Potent+inhibition+of+microRNA+in+vivo+without+degradation.">Potent inhibition of microRNA in vivo without degradation.</a> Nucleic Acids Research. 37 (1), 70-77 (2009).</li><li>Elmen, 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=LNA-mediated+microRNA+silencing+in+non-human+primates.">LNA-mediated microRNA silencing in non-human primates.</a> Nature. 452 (7189), 896-899 (2008).</li><li>Esau, C. 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Эта статья описал дизайн и синтез губкой Мирна и продемонстрировал, как ткани конкретным выражением Губка является мощным инструментом для ингибирования ткани специфические Мирна семьи выражение.
Мы продемонстрировали, что мир-181 семьи против Губка можно клонировать в ...

За последние два десятилетия были многочисленные исследования, которые указали на важную роль адаптивной в болезни человека. Выводы из большого количества литературы демонстрируют бесспорную важность интерферирующим в патофизиологии таких заболеваний, как рак1 и сердечно-сосудистых заболеваний2,3,4,5. К примеру мир-21-upregulated во многих раках, что приводит к увеличению клеток и клеточного цикла распространения6. В инфекции гепатита С мир-122 играет важную роль в репликации вируса7, и было показано, что ингибирование мир-122 снижает вирусную нагрузку8. В гипертрофии сердца мир-212/132-upregulated в самом сердце и участвует в патологический фенотип9. Очевидное значение Даунрегуляция или функциональной ингибитирование upregulated miRNA предлагает возможности для терапевтически эксплуатации Мирна биологии в практически всех заболеваний.
Четыре члена семьи мир-181, мир 181a/b/c/d, находятся в трех местах геномной в геноме человека. Intronic региона некодирующих хост гена РНК (MIR181A1-HG) кодирует кластер мир-181-a/b-1. Intronic область NR6A1 ген кодирует мир-181-a/b-2. Мир-181-c/d кластер расположен в uncharacterized транскрипт на хромосоме 19. Все члены семьи мир-181 поделиться той же последовательности «семян» и все четыре члена семьи мир-181 потенциально может регулировать же мРНК-мишеней.
Мы3,4 и другие10 высветили важность мир-181 членов семьи в течение последней стадии сердечной недостаточности. Мы также признали, что мир - 181c upregulation происходит в патологических условиях, связанных с повышенным риском сердечно-сосудистых заболеваний, например диабета II типа, ожирение и старения3,4,5. Постулируется, что гиперэкспрессия мир - 181c вызывает оксидативного стресса, что приводит к дисфункции сердца4.
Несколько групп предложили что Мирна существуют в митохондриях11,12,13,14, но мы были первыми, чтобы продемонстрировать, что мир - 181 c является производным от ядерного генома, обрабатываются, и впоследствии арестовано митохондрий в RISC3. Кроме того мы обнаружили выражение низкий мир 181a и мир 181b в отсеке митохондриальной сердца5. Важно отметить, что мы нашли что мир - 181 c подавляет выражение mRNA mt-COX1, так, продемонстрировав тем самым интерферирующим участвуют в регуляции митохондриальных генов и изменить функции митохондрий3,4.
Эта статья обсуждает методологии, необходимых для разработки Мирна губкой, чтобы постучать вниз весь мир-181 семьи в кардиомиоцитов. Кроме того мы приводим протокол для применения в естественных условиях мир-181-Губка.

<table>
	<tbody>
		<tr>
			<td>pEGFP-C1 vector</td>
			<td>Addgene</td>
			<td>6084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>In-fusion</td>
			<td>Clontech</td>
			<td>121416</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Miniprep</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>miR-181-sponge synthesis</td>
			<td>Introgen GeneArt</td>
			<td>custome made</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td>custome</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI enzymes</td>
			<td>New Endland Biolabs</td>
			<td>R0101S</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI enzymes</td>
			<td>New Endland Biolabs</td>
			<td>R0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid DNA Ligation Kit</td>
			<td>Sigma-Aldrich</td>
			<td>11635379001</td>
			<td></td>
		</tr>
		<tr>
			<td>H9c2 cells</td>
			<td>ATCC</td>
			<td>CRL-1446</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082139</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector 2b Device</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector Kits for H9c2 (2-1)</td>
			<td>Lonza</td>
			<td>VCA-1005</td>
			<td></td>
		</tr>
		<tr>
			<td>G418, Geneticin</td>
			<td>Thermo Fisher Scientific</td>
			<td>11811023</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria II Flow cytometer</td>
			<td>BD Bioscience</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 450 sonifier</td>
			<td>Marshall Scientific</td>
			<td>EDP 100-214-239</td>
			<td></td>
		</tr>
		<tr>
			<td>The Xenogen IVIS Spectrum optical imaging device</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MTCO1 antibody</td>
			<td>Abcam</td>
			<td>ab14705</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab7291</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequoia C256 ultrasound system</td>
			<td>Siemens</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo nanovector delivery of a heart-specific microrna-sponge

МикроРНК (miRNA) небольшой некодирующих РНК, которая препятствует выражение post-transcriptional матричная РНК (мРНК). Заболеваний человека, таких как рак и сердечно-сосудистых заболеваний, было показано, чтобы активировать ткани или клетки конкретных Мирна выражение, связанное с прогрессирования заболевания. Ингибирование Мирна выражения предлагает возможности для терапевтического вмешательства. Однако традиционные подходы к препятствовать адаптивной, используя antagomir олигонуклеотиды, влияют на функции конкретных Мирна после глобальной доставки. Здесь мы представляем протокол для ингибирования кардио конкретных в естественных условиях , мир-181 семьи в мышиной модели. Мирна Губка конструкция предназначена для включают 10 повторной привязки miR-181 последовательности. Кардио конкретных α-MHC промоутер клонируется в pEGFP позвоночника диск кардио конкретных мир-181 Мирна Губка выражение. Для создания стабильного ячейки линии выражая мир-181-Губка, myoblast H9c2 клетки являются transfected с α-MHC-EGFP-miR-181-sponge конструкцией и отсортированных по активации флуоресценции клеток, сортируя (FACs) в GFP положительные H9c2 клетки, которые культивировали с неомицин (G418). После стабильный рост в неомицин моноклональных клеточных популяций устанавливаются дополнительные СУИМ и одноклеточного клонирования. Результате клетки myoblast H9c2-мир-181-Губка GFP экспонат потеря функции членов семьи мир-181, как оценивать через увеличение выражение мир-181 целевых белков и по сравнению с H9c2 клеток, выражая схватка нефункциональные губкой. Кроме того мы разрабатываем nanovector для системных доставки построим мир-181-Губка, комплексообразующие положительно заряженных липосомальных наночастиц и отрицательно заряженные мир-181-Губка плазмид. В естественных условиях изображений GFP показывает, что множественные инъекции хвост вен nanovector в течение трех недель способны содействовать значительным проявлением мир-181-Губка в кардио конкретным образом. Важно отметить, что потеря функции мир-181 наблюдается в тканях сердца, но не в функции почек или печени. Мирна Губка-это мощный метод для подавления Мирна ткани конкретного выражения. Вождение Мирна Губка выражение из ткани конкретной промоутера обеспечивает специфичности для ингибирования Мирна, которые могут ограничиваться целевых органа или ткани. Кроме того сочетание nanovector и Мирна Губка технологий позволяет эффективной доставки и ткани конкретных Мирна ингибирование в естественных условиях.

В стабильно transfected pEGFP мир-181-Губка выражая H9c2 клеток (от шага 4.2) выражение всей семьи мир-181 (мир 181a, мир 181b, мир - 181c и мир - 181d) умеренно снизился по отношению к pEGFP яичница выражая H9c2 клеток. Мир-181-Губка служит конкурентный ингибитор весь мир-181 семьи, поэтому мы полагали, чт...

Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

В естественных условиях Nanovector поставка сердца конкретных микроРНК Губка

Ингибирование ткани конкретных микроРНК — это технология, развита в поле микроРНК. Здесь мы описываем протокол успешно подавляют микроРНК miR-181 семьи в myoblast клетки от сердца. Nanovector технология используется для доставки микроРНК Губка, которая демонстрирует значительный в vivo кардио конкретных мир-181 семьи ингибирование.

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

So for the last couple of decades, the importance of microRNA has been recognized in human disease conditions, but the problem is that, therapy-wise, it did not go to the primetime. One of the problem is, one microRNA can have different targets in different tissues. So one microRNA can be protected in one organ but detrimental to the others.So this method can help to eliminate tissue-specific microRNA knockdown strategy. The main advantage of this technology is we will use a microRNA sponge delivery and can be performed a tissue-specific manner. Begin this procedure with sponge design as described in the text protocol.To remove the CMV promoter from the commercially-available EGFR plasmid, first perform a digestion reaction. Following digestion, add five times the reaction volume of column binding buffer. Purify the digested linearized plasmid with a DNA purification column as described by the manufacturer.Elute with 25 microliters of elution buffer added carefully to the center column. Gel-purify the digested plasmid on a 0.5%agarose gel by first adding five microliters of loading buffer to the eluded plasmid. Load the entire sample into one well on a 0.5%agarose gel.Include a separate lane with 500 nanograms of uncut vector. Run it for 30 to 40 minutes, until an adequate separation of cut and uncut plasmids is achieved. After cloning the cardio-specific promoter alpha-myosin heavy chain as subscribed in the text protocol, add five times the reaction volume of the column binding buffer to the PCR reaction and purify it with a DNA purification column as described by the manufacturer.Elute the mixture with 25 microliters of elution buffer added carefully to the center of the column. Add five microliters of loading buffer to the eluded plasmid and load the entire sample into one well of a 1%agarose gel. Run the gel for 30 to 40 minutes before excising the PCR product as before.Ligate the alpha-myocin heavy chain promoter with the EGFP plasmid at 50 degrees Celsius for 15 minutes before cooling the reaction on ice. Perform a bacterial transformation by transfecting 50 microliters of competent cells with 2.5 microliters of infusion ligation mix. Rest the Eppendorf tube on ice for 20 minutes.Then, incubate the reaction in a 42 degree Celsius water bath for 40 seconds and place the tube on ice for two minutes. Following the transformation, add 350 microliters of SOC media and grow the cells at 37 degrees Celsius for 45 minutes. Plate 200 microliters of the outgrowth solution on LB plates with Kanamycin.Perform a colony PCR to identify Kanamycin-resistant cells, which contain the alpha-myosin heavy chain promoter ligation. Ligate the microRNA 181 sponge into the alpha-myosin heavy chain expressed EGFP vector by setting up a 21 microliter ligation reaction as detailed in the text protocol. Perform the ligation at room temperature for five minutes and then place it on ice.Transform 50 microliters of XL1-Blue competent cells with two microliters of ligation mix as before. Perform a colony PCR to identify Kanamycin-resistant bacteria, which contain the correct plasmid cloned with the first alpha-myacin heavy chain followed by the microRNA 181 sponge construct sequence. Design screening primers to PCR across the ligation junction.Expand the PCR positive clones in LB Kanamycin broth and in plasmids purified using standard mini-prep plasmid purification protocols. Perform DNA sequencing to verify that the plasmid is expressing the desired DNA sequences. Using an electroporator, transfect rat myoblasts with either a scramble sponge as a control, or the plasmid cloned with the first alpha-myocin heavy chain, followed by the microRNA 181 sponge construct.Briefly, transfect 0.4 million cells with two microgams of plasmid DNA and 100 microliters of a solution compatible with the electroporation device. Use an appropriate program to transfect the H9C2 cells. Plate only the GFP-expressed cells into a six-well plate with an addition of neomycin to the complete growth media.After seeding five to 10 cells in each well of a 96-well plate, followed by flow sorting, expand the monoclonal cells from the 96-well plate to 24-well plates over several passages. Prepare the liposomal nanoparticles by dissolving the components listed in the text protocol in a mixture of chloroform and methanol in a glass vial. Prepare the mixture by adding 5%glucose to the vacuum-dried lipid film before leaving it overnight to allow this mixture to hydrate the film.Gently rock the vial for two to three minutes at room temperature in order to produce multilamellar vesicles. Then, incubate the vesicles with occasional shaking in a 45 degree Celsius incubator. Sonicate the multilamellar vesicles to prepare small unilamellar vesicles in an ice bath for three to four minutes, until clarity can be observed.Add a 100%duty cycle and 25 watts of output power. Mix either a scramble sequence or a microRNA 181 sponge sequence cloned into the alpha-myocin heavy chain expressed EGFP plasmid with liposome on a one-to-three charge ratio basis to form the nanovector. The results of optimized three-week treatment protocols with six intravenous injections through the tail vein are shown here.The yellow colorization in the epifluorescence image demonstrates expression of the EGFP vector. The maximum intensity of yellow colorization is observed at the week three time point. The alpha-myosin heavy chain promoter sequence in the EGFP vector selectively expresses either microRNA 181 sponge or scrambled sequence in the heart at Day 21 as revealed by the yellow fluorescence.The Western blot and band densitometry showed a significant increase in the mt-COX1 expression in the microRNA 181 sponge nanovector injected rats as compared to the scramble nanovector groups, with alpha-Tubulin as the normalization control. One of the major challenges in this particular protocol is to keep the orientation of the sequencing, you need directional. Once a bacterial preparation is made, with a proper orientation of the microRNA sponge, this technique can be done with a two or three minute tail vein injection time.While attempting this procedure, it is important to sequence your bacterial colony multiple times, multiple batches, to make sure the microRNA response sequence is in the correct order. After watching this video, you should have a good understanding of how to design a microRNA sponge to knock down the expression of any microRNA family and prepare an in vivo application of microRNA sponge. Following this procedure, other methods, such as in vivo antagomir treatment can be performed to answer additional questions like whether a specific microRNA can be knocked down in all the organ at the same time.After its development, this particular technology paved the way for researcher in the field of microRNA to explore tissue-specific knockdown of a microRNA in the pathophysiology of multiple-disease conditions. Tissue-specific microRNA inhibition is currently an undeveloped technique in the microRNA field. The importance of mat-down regulation of functional inhibition of up-regulated microRNAs in human disease suggest exploring microRNA biology to create a cellular vulnerability for therapeutic intervention.

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Biology

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

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In vivo Imaging of Deep Cortical Layers using a Microprism

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Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

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RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

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Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

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Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

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Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

In Vivo Surface Electrocardiography for Adult Zebrafish

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The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

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Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving ...