Name: nanoparticle-mediated sirna gene-silencing in adult zebrafish heart
Uploaded: 2018-07-29T14:00:00
Description: Watch this Scientific Journal Video about Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart at JoVE.com

The authors thank Dr. IC Bruce for critical comments and reading the manuscript. This work was supported by grants from the National Natural Science Foundation of China (31430059, 31701272, 31730061, 81470399, and 31521062), AstraZeneca Asia, and Emerging Market Innovative Medicine and Early Development.

All animal procedures used a zebrafish protocol approved by the Institutional Animal Care and Use Committee at Peking University, which is fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care.
1. Preparation of Tricaine Solution
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	<li>To prepare tricaine stock solution, add 400 mg ethyl 3-aminobenzoate methanesulfonate powder to 97.9 mL distilled water, and then add 2.1 mL of 1 M Tris (pH 9.5) to adjust the pH to ~7. Store the stock solution at 4 &#1...

The zebrafish is fully capable of regenerating a variety of organs including the adult heart5. While transgenic and genetic methods are well-developed for studying gene functions in the embryos of zebrafish, investigators are still faced with the daunting task of generating conditional mutant alleles in zebrafish45,46. Thus, transgenic dominant-negative mutants or small-molecule inhibitors are frequently used to address gene functions in a...

Myocardial infarction has become a major health threat, leading to a tremendous economic burden around the world1. The adult mammalian heart fails to regenerate and replenish the lost cardiomyocytes on a macroscopic scale after the injury, leading to the formation of scar tissues and subsequent heart failure. Unlike mammals, the zebrafish is capable of heart regeneration, primarily through the robust myocardial proliferation after different types of heart injury, making it an ideal model organism for investigating the molecular mechanisms of heart regeneration2,3,4,5,6,7,8. Deciphering the endogenous mechanisms underlying zebrafish heart regeneration is an exciting area of research in the search for novel therapeutic strategies to improve human heart regeneration9.
Genetic manipulation methods are available in zebrafish. These consist of morpholinos (MO) that are also widely used in frogs, chick, and mammals besides in zebrafish10,11,12,13. MO has efficient knockdown of target gene expression in the adult zebrafish fin, brain, and retina14,15,16,17,18,19. Locked-nucleic acid (LNA) is another artificial oligonucleotide used to knock down endogenous gene expression not only in zebrafish embryos but also in adult animal organs20,21,22,23,24. However, the lack of effective loss-of-function methods for adults&#39; hearts remains an obstacle in studying the molecular mechanisms of organ regeneration. At present, small-molecule inhibitors or transgenic expression of dominant-negative mutants are primarily used to block the function of a certain gene or pathway to study its function in the adult zebrafish heart regeneration25,26,27. However, not all genes or signaling pathways are applicable for these methods.
Small-interfering RNAs (siRNAs) are widely used for the loss-of-function analysis in mammalian cells and embryos of model organisms, as well as adult organs for preclinical studies in animal models28,29,30,31,32. siRNAs have been effectively used to silence genes in tumors33,34,35 and in cardiomyocytes36,37,38,39,40&#160;via different delivery systems. Recently, we developed efficient siRNA-encapsulated nanoparticle gene-silencing in the regenerating adult heart using several different nanoparticles41,42,43, providing a novel tool for functional studies of genes in adult zebrafish organs. Based on our previous studies41,42,43, here we present a simple, practical, yet powerful protocol for siRNA gene-silencing in the regenerating adult zebrafish heart using f-PAMAM-PEG-R9 dendrimers. Aldh1a2 (aldehyde dehydrogenase 1 family, member A2) gene was upregulated after zebrafish apex resection and ablation of Aldh1a2 blocked the cardiac regeneration44. Here we take aldh1a2 gene as an example to test the gene knockdown efficiency mediated by nanoparticle-encapsulated siRNA injection. This protocol contains a procedure for zebrafish heart resection, chemical synthesis of nanoparticles, and a delivery method on siRNA-encapsulated nanoparticles into adult zebrafish heart.

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			<td style="width:167px;">Store at 4&#176;C</td>
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			<td height="21" style="height:21px;width:225px;">iridectomy scissors</td>
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			<td height="42" style="height:42px;width:225px;">&#945;,&#969;-dipyridyl disulfido polyethylene glycol(Py-PEG-Py)</td>
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			<td height="42" style="height:42px;width:225px;">core of G4.0 polyamidoamine (PAMAM)</td>
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			<td height="42" style="height:42px;width:225px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
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			<td height="42" style="height:42px;width:225px;">tris(2-carboxyethyl)phosphine(TCEP)</td>
			<td style="width:180px;">Alfar Aesar</td>
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			<td>Causes severe skin burns and eye damage. Causes serious eye damage.</td>
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nanoparticle-mediated sirna gene-silencing in adult zebrafish heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

To determine the efficiency of the dendrimer-mediated siRNA delivery, we resected the apex of the ventricle of the zebrafish heart, then injected about 10 &#181;L of dendrimer only (mock group), Cy5-siRNA only (naked group), or f-PAMAM-PEG-R9 dendrimer-encapsulated Cy5-siRNA (Cy5-siRNA group) intrapleurally, respectively (Figure 2A-B). The fluorescence signal was detectable in the hearts injected with dendrimer-encapsulated Cy5-siRNA at 3, 24...

Watch this Scientific Journal Video about Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart at JoVE.com

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

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