Name: delivery of modified mrna in a myocardial infarction mouse model
Uploaded: 2020-06-11T12:30:00
Description: Watch this Scientific Journal Video about Delivery of Modified mRNA in a Myocardial Infarction Mouse Model at JoVE.com

The authors acknowledge Ann Anu Kurian&#160;for her help with this manuscript. This work was funded by a cardiology start-up grant awarded to the Zangi laboratory and also by NIH grant R01 HL142768-01

All animal procedures outlined here have been approved by Icahn School of Medicine at Mount Sinai Institutional Care and Use Committee.
1. Synthesis of modRNA
NOTE: The details of modRNA synthesis can be found in Kondrat et al.13.
<ol>
	<li>Order the plasmid templates (Table of Materials) and generate a clean PCR product to use as the mRNA template.</li>
	<li>Prepare the modRNAs by transcription in vitro with a customized r...

<ol>
	<li>Muruve, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+innate+immune+response+to+adenovirus+vectors.">The innate immune response to adenovirus vectors.</a> Human Gene Therapy. 15 (12), 1157-1166 (2004).</li><li>Donsante, 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=Observed+incidence+of+tumorigenesis+in+long-term+rodent+studies+of+rAAV+vectors.">Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors.</a> Gene Therapy. 8 (17), 1343-1346 (2001).</li><li>Calcedo, R., Wilson, 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=Humoral+Immune+Response+to+AAV.">Humoral Immune Response to AAV.</a> Frontiers in Immunology. 4, 341 (2013).</li><li>Diebold, S. 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=Nucleic+acid+agonists+for+Toll-like+receptor+7+are+defined+by+the+presence+of+uridine+ribonucleotides.">Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides.</a> European Journal of Immunology. 36 (12), 3256-3267 (2006).</li><li>Magadum, A., Kaur, K., Zangi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA-Based+Protein+Replacement+Therapy+for+the+Heart.">mRNA-Based Protein Replacement Therapy for the Heart.</a> Molecular Therapy. 27 (4), 785-793 (2019).</li><li>Kariko, K., 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=Incorporation+of+pseudouridine+into+mRNA+yields+superior+nonimmunogenic+vector+with+increased+translational+capacity+and+biological+stability.">Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.</a> Molecular Therapy. 16 (11), 1833-1840 (2008).</li><li>Hadas, Y., 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=Optimizing+Modified+mRNA+In+Vitro+Synthesis+Protocol+for+Heart+Gene+Therapy.">Optimizing Modified mRNA In Vitro Synthesis Protocol for Heart Gene Therapy.</a> Molecular Therapy- Methods and Clinical Development. 14, 300-305 (2019).</li><li>Sultana, 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=Optimizing+Cardiac+Delivery+of+Modified+mRNA.">Optimizing Cardiac Delivery of Modified mRNA.</a> Molecular Therapy. 25 (6), 1306-1315 (2017).</li><li>Zangi, 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=Modified+mRNA+directs+the+fate+of+heart+progenitor+cells+and+induces+vascular+regeneration+after+myocardial+infarction.">Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.</a> Nature Biotechnology. 31 (10), 898-907 (2013).</li><li>Carlsson, 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=Purified+VEGF-A+mRNA+Improves+Cardiac+Function+after+Intracardiac+Injection+1+Week+Post-myocardial+Infarction+in+Swine.">Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection 1 Week Post-myocardial Infarction in Swine.</a> Molecular Therapy Methods Clinical Development. 9, 330-346 (2018).</li><li>Huang, C. 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=Synthetic+chemically+modified+mRNA-based+delivery+of+cytoprotective+factor+promotes+early+cardiomyocyte+survival+post-acute+myocardial+infarction.">Synthetic chemically modified mRNA-based delivery of cytoprotective factor promotes early cardiomyocyte survival post-acute myocardial infarction.</a> Molecular Pharmaceutics. 12 (3), 991-996 (2015).</li><li>Magadum, 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=Ablation+of+a+Single+N-Glycosylation+Site+in+Human+FSTL+1+Induces+Cardiomyocyte+Proliferation+and+Cardiac+Regeneration.">Ablation of a Single N-Glycosylation Site in Human FSTL 1 Induces Cardiomyocyte Proliferation and Cardiac Regeneration.</a> Molecular Therapy - Nucleic Acids. 13, 133-143 (2018).</li><li>Kondrat, J., Sultana, N., Zangi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+Modified+mRNA+for+Myocardial+Delivery.">Synthesis of Modified mRNA for Myocardial Delivery.</a> Methods in Molecular Biology. 1521, 127-138 (2017).</li><li>Gan, L. 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=Intradermal+delivery+of+modified+mRNA+encoding+VEGF-A+in+patients+with+type+2+diabetes.">Intradermal delivery of modified mRNA encoding VEGF-A in patients with type 2 diabetes.</a> Nature Communication. 10 (1), 871 (2019).</li></ol>

Gene therapy has shown tremendous potential to significantly advance the treatment of cardiac disease. However, traditional tools employed in the initial clinical trials for treatment of HF have shown limited success and are associated with severe side effects. Modified RNA presents a nonviral gene delivery that is continuously gaining popularity as a gene transfer tool in the heart. ModRNA requires no nuclear localization of genes for translation, and thus offers an efficient and fast expression of the protein. Further,...

Gene therapy is a powerful tool involving delivery of nucleic acids for the treatment, cure, or prevention of human diseases. Despite the progress in the diagnostic and therapeutic approaches for heart disease, there has been limited success in the delivery of genes in myocardial infarction (MI) and heart failure (HF). As straightforward as the process of gene therapy seems, it is a markedly complex approach considering the many factors that need to be optimized before employing a particular delivery vehicle. The correct delivery vector should be non-immunogenic, efficient, and stable inside the human body. Efforts in this field have generated two types of delivery systems: viral or non-viral. The widely used viral systems, including gene transfer by adenovirus, retrovirus, lentivirus, or adeno-associated virus, have shown exceptional transduction capacity. However, their use in clinics is limited due to the strong immune response induced1, risk of tumorigenesis2, or the presence of neutralizing antibodies3, all of which remain a major obstacle to broad and effective application of viral vectors in human gene therapy. On the other hand, despite their impressive expression pattern, the delivery of naked plasmid DNA displays a low transfection efficiency, while mRNA transfer presents high immunogenicity and susceptibility to degradation by RNase4.
With the extensive research in the field of mRNA, modRNA has become an attractive tool for delivery of genes to the heart and various other organs due to its numerous advantages over traditional vectors5. Complete replacement of uridine with naturally occurring pseudouridine results in more robust and transient protein expression, with minimal induction of innate immune response and risk of genomic integration6. Recently established protocols use an optimized amount of anti-reverse cap analog (ARCA) that further enhances the protein translation by increasing the stability and transability of the synthetic mRNA7.
Previous reports have shown the expression of various reporter or functional genes delivered by modRNA in the rodent myocardium after MI. With modRNA applications, significant areas of the myocardium, including both cardiomyocytes and noncardiomyocytes, have been successfully transfected post-cardiac injury8 to induce angiogenesis9,10, cardiac cell survival11, and cardiomyocyte proliferation12. A single administration of modRNA encoded for mutated human follistatin-like 1 induces the proliferation of mouse adult CMs and significantly increases cardiac function, decreases scar size, and increases capillary density 4 weeks post-MI12. A more recent study reported improved cardiac function after MI with application of VEGFA modRNA in a swine model10.
Thus, with the increased popularity of modRNA in the cardiac field, it is essential to develop and optimize a protocol for the delivery of modRNA to the heart post-MI. Herein is a protocol describing the preparation and delivery of purified and optimized modRNA in a biocompatible citrate-saline formulation that provides robust, stable protein expression without stimulating any immune response. The method shown in this protocol and video demonstrates the standard surgical procedure of a mouse MI by permanent ligation of the left anterior descending artery (LAD), followed by three site intracardiac injections of modRNA. The aim for this paper is to clearly define a highly accurate and reproducible method of modRNA delivery to the murine myocardium to make modRNA application widely accessible for cardiac gene therapy.

<table>
	<tbody>
		<tr>
			<td>Adenosine triphosphate</td>
			<td>Invitrogen</td>
			<td>AMB13345</td>
			<td>Included in Megascript kit</td>
		</tr>
		<tr>
			<td>Antarctic Phosphatase</td>
			<td>New England Biolabs</td>
			<td>M0289L</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-reverse cap analog, 30-O-Mem7G(50) ppp(50)G</td>
			<td>TriLink Biotechnologies</td>
			<td>N-7003</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioluminescense imaging system</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>IVIS100 charge-coupled device imaging system</td>
		</tr>
		<tr>
			<td>Blunt retractors</td>
			<td>FST</td>
			<td>18200-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac tropnin I</td>
			<td>Abcam</td>
			<td>47003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytidine triphosphate</td>
			<td>Invitrogen</td>
			<td>AMB13345</td>
			<td>Included in Megascript kit</td>
		</tr>
		<tr>
			<td>Dual Anesthesia System</td>
			<td>Harvard Apparatus</td>
			<td>75-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps- Adson</td>
			<td>FST</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps- Dumont #7</td>
			<td>FST</td>
			<td>91197-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine triphosphate</td>
			<td>Invitrogen</td>
			<td>AMB13345</td>
			<td>Included in Megascript kit</td>
		</tr>
		<tr>
			<td>In vitro transcription kit</td>
			<td>Invitrogen</td>
			<td>AMB13345</td>
			<td>5X MEGAscript T7 Kit</td>
		</tr>
		<tr>
			<td>Intubation cannula</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Megaclear kit</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse ventilator</td>
			<td>Harvard Apparatus</td>
			<td>73-4279</td>
			<td></td>
		</tr>
		<tr>
			<td>N1-methylpseudouridine-5-triphosphate</td>
			<td>TriLink Biotechnologies</td>
			<td>N-1081</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop Spectrometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen hegar needle holder with suture scissors</td>
			<td>FST</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid templates</td>
			<td>GeneArt, Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp-Pointed Dissecting Scissors</td>
			<td>FST</td>
			<td>14200-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon</td>
			<td>Y433H</td>
			<td>5.00</td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon</td>
			<td>Y432H</td>
			<td>6.00</td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon</td>
			<td>7733G</td>
			<td>7.00</td>
		</tr>
		<tr>
			<td>T7 DNase enzyme</td>
			<td>Invitrogen</td>
			<td>AMB13345</td>
			<td>Included in Megascript kit</td>
		</tr>
		<tr>
			<td>Tape station</td>
			<td>Aligent</td>
			<td>4200</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcription clean up kit</td>
			<td>Invitrogen</td>
			<td>AM1908</td>
			<td>Megaclear</td>
		</tr>
		<tr>
			<td>Ultra-4 centrifugal filters 10k</td>
			<td>Amicon</td>
			<td>UFC801096</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of modified mrna in a myocardial infarction mouse model

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.

Eight to ten-week-old mice were anesthetized with isoflurane and intubated. After the animal was under anesthesia, the left thoracic region was shaved and sterilized with ethanol, and the heart was exposed for LAD ligation. The left coronary artery was occluded by firmly knotting the suture under the artery (diagram representation Figure 1A). After a successful infarction (indicated by the paling of the left ventricular free wall), a direct injection of 100 &#181;g of Luc or Cre modRNA disso...

Watch this Scientific Journal Video about Delivery of Modified mRNA in a Myocardial Infarction Mouse Model at JoVE.com

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

This protocol presents a simple and coherent way to transiently upregulate a gene of interest using modRNA after myocardial infarction in mice.

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising ...

Modified RNA is a novel method for gene transfer to the heart, and other organs. This protocol will provide accurate approach of delivering desired genes transiently to the heart, post myocardial infarction. This method is not limited to myocardial infarction, but can also be applied to ischemia reperfusion, and transverse aortic constriction models.Modified RNA is being used in several human clinical trials. However, there is no standardized protocol for gene transferred into the heart using this method. Visual demonstration will enable researchers to correctly inject modified RNA at appropriate sites in the heart.Demonstrating the procedule will be Ajit Magadum, a postdoctoral fellow, and Elena Chepurko an animal supervisor from our laboratory. Based on the concentration of the previously prepared at modRNA, calculate the volume of modRNA needed for a 100 microgram injection. Makes equal volumes of sucrose and citrate solutions.Then add the calculated volume of modRNA, and enough saline solution to bring the injection volume to 60 microliters. After anesthetizing the animal, place it ventral side up, with its mouth facing toward the experimenter. Then to maintain an open airway by performing intratracheal intubation, gently pull out the tongue.Adjust the angle of the neck to straighten the intubation path, and push in the intubation cannula. Observe the thoracic movement of the mouse to verify that both lungs are receiving oxygen. The respiratory rate should be approximately 120 breaths per minute.To begin the LAD ligation, turn the mouse carefully onto its right side. lift the skin, and perform a left-sided thoracotomy between the third and fourth ribs. Open the thorax carefully, without touching the lungs with any sharp objects.Place retractors into the incision to keep the thoracic cavity open, providing a clear view of the heart. Next, move the lungs to the edge of the incision, and remove the part of the pericardial sac that is covering the heart. Carefully identify the LAD, a deep light red vessel, located between the pulmonary artery and the left auricle.Ligate the proximal LAD with a single silk suture. Using an insulin syringe, deliver a total of 60 microliters of the modRNA solution to the heart. Inject 20 microliters intramuscularly, at three different sites.One site on each side of the ligation and one in the apex of the heart. The best way to inject the modified RNA in the correct sites, is to observe the discoloration of the tissue under microscope, after the occlusion of LAD. Also when injected, do not go too deep into the tissue to reach the heart chambers into the circulation.Close the thoracic incision in layers. First use was 6-0 silk running sutures to adapt the ribs. Then use 5-0 silk running sutures to close the skin.For the recovery phase, remove the cannula, and allow spontaneous breathing. Place the animal on a heating pad until it regains consciousness. Do not leave the animal unattended until it is fully awake.To manage postsurgical pain, inject buprenorphine subcutaneously at 12 hour intervals for three days. At a dose of 0.1 milligrams per kilogram of body weight. 24 hours after LAD ligation and injection of luciferase modRNA in CFW mice, transfection was validated by checking for luciferase protein expression using a bioluminescence imaging system.The luciferase signal can be clearly detected in images comparing control mice, to mice injected with luciferase modRNA. Quantification of the luciferase signal shows significantly higher protein expression in treated mice than in control mice. Cre modRNA expression was validated by checking its translation and bio distribution in transgenic ROSA26 mTmG mice.24 hours after surgery and cre modRNA injection, arts were fixed and processed for immunostaining with cardiomyocyte marker, cardiac troponin I, and nuclei marker DAPI. Successful cre expression was evident due to the appearance of green colored cardiomyocytes. A merged image shows cre activated cells around the two visible injection sites.Blue is the nuclear stain DAPI. Modified RNA platform can deliver a single gene or gene combinations in variety of organs. And thus can be used for therapeutic purposes, such as correcting genetic disorders, or activating beneficial mechanisms in the body.The unique pharmacokinetics of modified RNA enables us to evaluate gene function immediately after its injection. This enables researcher to target signaling pathways, which are altered in the first few hours of infarction.

delivery-of-modified-mrna-in-a-myocardial-infarction-mouse-model

Research

JoVE Journal

Genetics

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

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

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

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

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

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

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is pathogenic, one must query many databases to determine whether the gene of interest is linked to a genetic disease, whether the specific variant has been reported before, and what functional data is available in model organism databases that may provide clues about the gene&#8217;s function in human. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) is a one-stop data collection tool for human genes and variants and their orthologous genes in seven model organisms including in mouse, rat, zebrafish, fruit fly, nematode worm, fission yeast, and budding yeast. In this Protocol, we provide an overview of what MARRVEL can be used for and discuss how different datasets can be used to assess whether a variant of unknown significance (VUS) in a known disease-causing gene or a variant in a gene of uncertain significance (GUS) may be pathogenic. This protocol will guide a user through searching multiple human databases simultaneously starting with a human gene with or without a variant of interest. We also discuss how to utilize data from OMIM, ExAC/gnomAD, ClinVar, Geno2MP, DGV and DECHIPHER. Moreover, we illustrate how to interpret a list of ortholog candidate genes, expression patterns, and GO terms in model organisms associated with each human gene. Furthermore, we discuss the value protein structural domain annotations provided and explain how to use the multiple species protein alignment feature to assess whether a variant of interest affects an evolutionarily conserved domain or amino acid. Finally, we will discuss three different use-cases of this website. MARRVEL is an easily accessible open access website designed for both clinical and basic researchers and serves as a starting point to design experiments for functional studies.

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...