Name: Författare i fokus: Hjärtcellstransgenes för snabb genscreening
Uploaded: 2024-05-24T14:25:00
Description: The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

Denna studie stöddes av anslag RTI2018-097617-J-I00 från det spanska Ministerio de Ciencia e Innovación och Acción 9 från Universidad de Jaén till O.H.O. Grant PGC2018-096486-B-I00 från det spanska Ministerio de Ciencia e Innovación och bidrag H2020-MSCA-ITN-2016-722427 från EU:s Horizon 2020-program till M.T. JMG stöddes av ett doktorandstipendium från det spanska vetenskapsministeriet och Fundación Severo Ochoa (PRE2022-101884). Både CNIC och CBMSO stöds av det spanska vetenskapsministeriet, och CNIC stöds av ProCNIC Foundation.

Alla djurförsök godkändes av CNIC Animal Experimentation Ethics Committee och överensstämde med gällande lagstiftning, inklusive EU-direktiv 2010/63EU och rekommendation 2007/526/EG, som verkställs av spansk lag enligt Real Decreto 53/2013. För detta protokoll användes honmöss av vildtyp CD-1 i åldern 15-21 veckor. Detaljer om de djur, reagenser och utrustning som används finns i materialtabellen.
1. Förberedelse av plasmid och verktyg
<ol><li>B...

Elektroporationsbaserad transgenes av hjärtceller i mushjärtan

<ol>
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V., 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=Characterization+of+a+common+progenitor+pool+of+the+epicardium+and+myocardium.">Characterization of a common progenitor pool of the epicardium and myocardium.</a> Science. 371 (6533), eabb2986 (2021).</li><li>Sendra, M., Dom&#237;nguez, J., Torres, M., Oca&#241;a, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+complexity+of+early+heart+progenitor+cells.">Dissecting the complexity of early heart progenitor cells.</a> J Cardiovasc Dev Dis. 9 (1), 5 (2021).</li><li>Ivanovitch, K., Temi&#241;o, S., Torres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+heart+tube+development+in+mouse+reveals+alternating+phases+of+cardiac+differentiation+and+morphogenesis.">Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis.</a> eLife. 6, e30668 (2017).</li><li>Ai, 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=Canonical+Wnt+signaling+functions+in+second+heart+field+to+promote+right+ventricular+growth.">Canonical Wnt signaling functions in second heart field to promote right ventricular growth.</a> PNAS. 104 (22), 9319-9324 (2007).</li><li>Zimmerman, M. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration+from+activated+epicardium.">Cardiac regeneration from activated epicardium.</a> PLOS One. 7 (9), e44692 (2012).</li><li>Hesse, 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=Single-cell+transcriptomics+defines+heterogeneity+of+epicardial+cells+and+fibroblasts+within+the+infarcted+murine+heart.">Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart.</a> eLife. 10, e65921 (2021).</li><li>Streef, T. J., Smits, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+contribution+to+the+developing+and+injured+heart:+Exploring+the+Cellular+composition+of+the+epicardium.">Epicardial contribution to the developing and injured heart: Exploring the Cellular composition of the epicardium.</a> Front Cardiovasc Med. 8, 750243 (2021).</li><li>Sanchez-Fernandez, 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=Understanding+epicardial+cell+heterogeneity+during+cardiogenesis+and+heart+regeneration.">Understanding epicardial cell heterogeneity during cardiogenesis and heart regeneration.</a> J Cardiovasc Dev Dis. 10 (9), 376 (2023).</li><li>Quijada, 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=Coordination+of+endothelial+cell+positioning+and+fate+specification+by+the+epicardium.">Coordination of endothelial cell positioning and fate specification by the epicardium.</a> Nat Commun. 12 (1), 4155 (2021).</li><li>Mantri, 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=Spatiotemporal+single-cell+RNA+sequencing+of+developing+chicken+hearts+identifies+interplay+between+cellular+differentiation+and+morphogenesis.">Spatiotemporal single-cell RNA sequencing of developing chicken hearts identifies interplay between cellular differentiation and morphogenesis.</a> Nat Commun. 12 (1), 1771 (2021).</li><li>Paul, S., Zhang, X., He, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeobox+gene+Meis1+modulates+cardiovascular+regeneration.">Homeobox gene Meis1 modulates cardiovascular regeneration.</a> Semin Cell Dev Biol. 100, 52-61 (2020).</li><li>Stankunas, 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=Pbx/Meis+deficiencies+demonstrate+multigenetic+origins+of+congenital+heart+disease.">Pbx/Meis deficiencies demonstrate multigenetic origins of congenital heart disease.</a> Circ Res. 103 (7), 702-709 (2008).</li><li>Liu, 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=Transcription+factor+Meis1+act+as+a+new+regulator+of+ischemic+arrhythmias+in+mice.">Transcription factor Meis1 act as a new regulator of ischemic arrhythmias in mice.</a> J Adv Res. 39, 275-289 (2022).</li><li>Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: A laboratory manual. , (2014).</li><li>Wong, M. 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A., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+myosins:+expression+patterns+and+functional+significance.">Developmental myosins: expression patterns and functional significance.</a> Skelet. Muscle. 5 (1), 22 (2015).</li><li>Eissa, 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=Stability+of+reference+genes+for+messenger+RNA+quantification+by+real-time+pcr+in+mouse+dextran+sodium+sulfate+experimental+colitis.">Stability of reference genes for messenger RNA quantification by real-time pcr in mouse dextran sodium sulfate experimental colitis.</a> PLOS One. 11 (5), e0156289 (2016).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2&#8722;&#916;&#916;CT+method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2&#8722;&#916;&#916;CT method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Lai, S. R., Andrews, L. G., Tollefsbol, T. O. <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+using+a+plasmid+construct+expressing+short-hairpin+RNA.">RNA interference using a plasmid construct expressing short-hairpin RNA.</a> Methods Mol Biol. 405, 31-37 (2007).</li><li>Carmona, R., Barrena, S., L&#243;pez Gambero, A. 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Sammantaget erbjuder den metod som beskrivs här ett robust ramverk för att uttrycka transgena konstruktioner i det utvecklande epikardiet (Figur 4B), vilket demonstreras av Meis1-överuttryck (Figur 4C). Med lämpliga konstruktioner kan detta protokoll användas för att tillfälligt bedöma effekten av antingen gain-of-function (GOF) eller loss-of-function (LOF) av en specifik gen. LOF kan implementeras i tekniken genom att transfektera en plasmid riktad mot ...

Hjärtat är det första organ som bildas under den embryonala utvecklingen. Denna process involverar den spatiotemporala samordningen av olika populationer av stamceller från distinkta områden av embryot. Allt detta sker medan det växande hjärtat fortsätter att slå och fungera, vilket understryker den anmärkningsvärda koordination som krävs för attdet ska kunna bildas. Med tanke på hjärtats avgörande roll är det viktigt med en tät reglering på cellulär och molekylär nivå för att det ska bildaspå rätt sätt. Att identifiera de mekanismer som styr hjärtats utveckling har varit av stort intresse, eftersom de är avgörande för att reda ut medfödda hjärtsjukdomar, som drabbar ett stort antal patienter över hela världen6. Dessutom är det avgörande att förstå hjärtats utveckling för att dechiffrera hjärtats regenerering, eftersom postnatala däggdjurshjärtan behåller en regenerativ kapacitet som förloras eller hindras i vuxen ålder 7,8. Följaktligen är det absolut nödvändigt att dissekera molekylära regulatorer för hjärtats utveckling för att främja forskningsinsatser om medfödda hjärtsjukdomar och hjärtregenerering.
I strävan efter detta mål har det funnits ett växande fokus på att undersöka epikardiets roll i hjärtats utveckling och regenerering9. Epikardiet är ett tunt lager av mesotelial vävnad som utgör det yttersta lagret av däggdjurshjärtat (figur 1). Nyligen genomförda studier har visat epikardiets betydelse vid hjärtskada, vilket avslöjar att denna vävnad kan skicka proliferationssignaler till kardiomyocyter i det drabbade området för att mildra skadan10,11. Trots epikardiets betydelse har det varit svårt att genomföra ytterligare molekylära undersökningar på grund av dess enorma heterogenitet. Encelliga RNAseq-experiment har avslöjat epikardiets heterogenitet, som hyser flera cellsubpopulationer med distinkta transkriptomiska signaturer 12,13,14,15,16. En strategi för att screena potentiella regulatorer för hjärtutveckling bör därför ta hänsyn till mångfalden av epikardiella stamceller.
I denna mening har musmodellens mottaglighet för genetisk modifiering underlättat identifieringen av många gener som är avgörande för hjärtats utveckling, vilket möjliggör generering av mutantlinjer med gain-of-function (GOF) eller loss-of-function (LOF) av specifika gener. Dessa tillvägagångssätt innebär dock en betydande investering av tid och experimentella resurser; Därför är de opraktiska när man bedömer rollerna för ett stort antal kandidatgener. Dessutom utövar utvecklingsgener ofta pleiotropa funktioner i olika vävnader eller krävs för tidig embryonal utveckling, vilket hindrar tolkningen av deras bidrag till utvecklingen i en specifik process. Även om det är möjligt att rikta in sig på genernas funktion vid specifika strukturer eller utvecklingstidpunkter, kräver detta vanligtvis användning av mer komplexa genetiska konstruktioner, som kan vara svåra att generera eller som i allmänhet inte är tillgängliga.
För att övervinna dessa begränsningar presenterar vi en metod för att elektroporera musembryonala hjärtan för transient transgenes (Figur 2). Tillsammans med ex vivo-odling och fluorescensaktiverad cellsortering (FACS) visar denna strategi sina möjligheter genom transient GOF av Meis1, en välkarakteriserad gen som är inblandad i hjärtutveckling och regenerering 17,18,19. I den här artikeln undersöks också andra potentiella tillämpningar av denna metod, och dess fördelar och begränsningar diskuteras, liksom jämförs med befintliga protokoll för transientt modulerande genuttryck. Vi tror att ramverket och de visuella exemplen som presenteras kommer att öka förståelsen för epikardiets biologi under utveckling och sjukdom.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66754/66754fig01v2.jpg"> Figur 1: Embryonala hjärtskikt hos möss. Schematisk bild av en koronal bild av ett E13-14 musembryonalt hjärta. Hjärtats tre huvudsakliga cellulära lager representeras i gult (endokardiet), rött (myokardium) och blått (epikardium). Hjärtsäcken är representerad med en brun linje. Hjärtats fyra kammare förkortas LV, vänster kammare; RV, höger kammare; LA, vänster förmak; RA, höger förmak. <a href="https://www.jove.com/files/ftp_upload/66754/66754fig01largev2.jpg" target="_blank">Klicka här för att se en större version av denna figur.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66754/66754fig02.jpg"> Figur 2: Schematisk översikt över hjärtelektroporationsprotokollet. <a href="https://www.jove.com/files/ftp_upload/66754/66754fig02large.jpg" target="_blank">Klicka här för att se en större version av denna figur.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#55 Forceps</td>
			<td>Dumont&#160;</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well Clear Flat Bottom Multiwell Cell Culture Plate</td>
			<td>BD Falcon</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm vise table&#160;</td>
			<td>Grandado</td>
			<td>SKU 8798771617573</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Fischer Scientific</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Cell Strainer</td>
			<td>Corning</td>
			<td>CLS431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP Policlonal Antibody</td>
			<td>Invitrogen</td>
			<td>A10262</td>
			<td>1:1000 dilution used</td>
		</tr>
		<tr>
			<td>Anti-Myosin 4 (MF20) Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>14-6503-82</td>
			<td>1:500 dilution used</td>
		</tr>
		<tr>
			<td>CD1 Wild Type mice</td>
			<td></td>
			<td></td>
			<td>Provided by Animalary Unit (CNIC)</td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 (Asp175) Antibody</td>
			<td>Cell Signalling Technologies</td>
			<td>9661</td>
			<td>1:400 dilution used</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Cell Signalling Technologies</td>
			<td>4083</td>
			<td>1:1000 dilution used</td>
		</tr>
		<tr>
			<td>Dispase/collagenase</td>
			<td>Roche</td>
			<td>10269638001</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>10313021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>10438-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51032720</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Stereoscopic Microscope S8AP0</td>
			<td>Leica</td>
			<td>11524102</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller Model P-97</td>
			<td>Sutter Instrument</td>
			<td>SU-P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG expression plasmid</td>
			<td>Addgene</td>
			<td>#89689</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 &#215; 10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 60 &#215; 15 mm</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Merck</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td></td>
			<td></td>
			<td>Reused from old laboratory equipment</td>
		</tr>
		<tr>
			<td>Rat Serum culture embryo, male rats SPRAGUE DAWLEY RjHan SD</td>
			<td>Janvier Labs</td>
			<td>9979</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant anti-Wilms Tumor Protein 1 (WT1) Antibody</td>
			<td>Abcam</td>
			<td>ab89901</td>
			<td>1:300 dilution used</td>
		</tr>
		<tr>
			<td>Square Wave Electroporator CUY21SC</td>
			<td>Nepa Gene</td>
			<td>CUY664-10X15</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td></td>
			<td></td>
			<td>Provided and autoclaved by technical unit</td>
		</tr>
		<tr>
			<td>Sucrose&#160;</td>
			<td>Millipore</td>
			<td>84100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer electrodes with variable gap</td>
			<td>Nepa Gene</td>
			<td>CUY650P5</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient transgenesis approach for gene delivery in the mouse embryonic heart

Däggdjurshjärtat är ett komplext organ som bildas under utvecklingen via mycket olika populationer av stamceller. Ursprunget, tidpunkten för rekryteringen och ödet för dessa förfäder är avgörande för en korrekt utveckling av detta organ. De molekylära mekanismer som styr hjärtats morfogenes är viktiga för att förstå patogenesen av medfödda hjärtsjukdomar och embryonal hjärtregenerering. Klassiska metoder för att undersöka dessa mekanismer använde generering av transgena möss för att bedöma funktionen av specifika gener under hjärtutvecklingen. Transgenes hos möss är dock en komplex och tidskrävande process som ofta inte kan utföras för att bedöma vilken roll specifika gener spelar under hjärtats utveckling. För att ta itu med detta har vi utvecklat ett protokoll för effektiv elektroporering och odling av musembryonala hjärtan, vilket möjliggör transient transgenes för att snabbt bedöma effekten av vinst eller förlust av funktion hos gener som är involverade i hjärtutveckling. Med hjälp av denna metodik överuttryckte vi framgångsrikt Meis1 i det embryonala hjärtat, med en preferens för epikardiell celltransfektion, vilket visar teknikens kapacitet.

The heart is the first organ formed during embryonic development. This process involves the spatiotemporal coordination of various populations of progenitor cells from distinct areas of the embryo. All this occurs while the developing heart continues to beat and function, emphasizing the remarkable coordination required for its formation1,2,3. Given the crucial role of the heart, tight regulation at the cellular and molecular levels is essential for its proper formation4,5. Identifying the mechanisms that control heart development has been of great interest, as they are crucial for unraveling congenital heart disorders, which impact a substantial number of patients worldwide6. Furthermore, comprehending heart development is pivotal in deciphering cardiac regeneration, as postnatal mammalian hearts retain a regenerative capacity that is lost or hindered in adulthood7,8. Consequently, dissecting molecular regulators of heart development is imperative to advance research efforts on congenital heart disease and cardiac regeneration.
In pursuit of this objective, there has been a growing focus on investigating the role of the epicardium in cardiac development and regeneration9. The epicardium is a thin layer of mesothelial tissue that comprises the outermost layer of the mammalian heart (Figure 1). Recent studies have shown the importance of the epicardium during cardiac injury, revealing that this tissue is able to send proliferation signals to cardiomyocytes in the affected area to mitigate the damage10,11. Despite the importance of the epicardium, conducting further molecular investigations has been challenged by its immense heterogeneity. Single-cell RNAseq experiments have revealed the epicardium&#39;s heterogeneity, housing multiple cell subpopulations with distinct transcriptomic signatures12,13,14,15,16. Thus, a strategy to screen potential regulators of cardiac development should accommodate the diversity of epicardial progenitor cells.
In this sense, the mouse model&#39;s amenability to genetic modification has facilitated the identification of numerous genes crucial for heart development, allowing the generation of mutant lines with gain-of-function (GOF) or loss-of-function (LOF) of specific genes. However, these approaches imply a considerable investment of time and experimental resources; therefore, they are impractical when assessing the roles of a large number of candidate genes. Besides, developmental genes often exert pleiotropic functions in different tissues or are required for early embryonic development, hampering the interpretation of their contribution to development in a specific process. While it is possible to target gene function at specific structures or developmental time points, this usually requires the use of more complex genetic constructions, which can be difficult to generate or are generally unavailable.
To overcome these limitations, we present a methodology to electroporate mouse embryonic hearts for transient transgenesis (Figure 2). Paired with ex vivo culture and fluorescence-activated cell sorting (FACS), this strategy demonstrates its capabilities through transient GOF of Meis1, a well-characterized gene implicated in heart development and regeneration17,18,19. In this article, other potential applications of this methodology are also explored, and its advantages and limitations are discussed, as well as compared to existing protocols for transiently modulating gene expression. We believe the framework and visual examples presented will enhance the understanding of epicardium biology during development and disease.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66754/66754fig01v2.jpg" /> 
Figure 1: Mouse embryonic heart layers. Schematic diagram of a coronal view of an E13-14 mouse embryonic heart. The three main cellular layers of the heart are represented in yellow (endocardium), red (myocardium), and blue (epicardium). The pericardium is represented in a brown line. The four chambers of the heart are abbreviated as LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium. <a href="https://www.jove.com/files/ftp_upload/66754/66754fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66754/66754fig02.jpg" /> 
Figure 2: Schematic overview of the heart electroporation protocol. <a href="https://www.jove.com/files/ftp_upload/66754/66754fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Författare i fokus: Hjärtcellstransgenes för snabb genscreening

En effektiv transgenesmetod för genleverans i musens embryonala hjärta

We aim to establish a new methodology that facilitates the overexpression or downregulation of a certain gene to understand heart development. Mouse transgenesis is a great investment of time and effort. Assessing genes one by one is not realistic because of this and also because of the high complexity of the genome.Hence, there is a need for a rapid method of screening. We have presented a tool available to any researcher to analyze the gain or loss of function of a candidate gene during heart development. The main advantage of our protocol is that it's much quicker and less expensive than mouse transgenesis.Perhaps it could be used to assess different genes of interest before commencing a transgenesis experiment.

För att demonstrera effektiviteten av denna teknik för att utföra gain-of-function-experiment (GOF) för relevanta hjärtutvecklingsregulatorer, elektroporerades en konstruktion som överuttryckte Meis1-transkriptionsfaktorn. För att uppnå detta extraherades RNA från E9.5-embryon och omvänd transkription utfördes för att erhålla komplementärt DNA (cDNA). Genom att använda cDNA som mall klonades Meis1-kodningssekvensen (tilläggstabell 1) till en pCAG-uttrycksplasmid (hädanef...

Detta protokoll presenterar ett detaljerat metodologiskt ramverk för elektroporationsbaserad transgenes av hjärtceller i mushjärtan under utveckling. Videotillgångarna som tillhandahålls här kommer att underlätta inlärningen av denna mångsidiga teknik.

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

an-efficient-transgenesis-approach-for-gene-delivery-mouse-embryonic

Research

JoVE Journal

Developmental Biology

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

Extraction and Electroporation of Mouse Embryo Heart as a Transgenesis Approach

To begin, use a pipette puller to stretch a 20 micrometer wide glass capillary. Place an extracted mouse uterus on a dry paper towel. Then, insert the tip of a pair of fine scissors into the myometrial side of the uterus, and slowly run the edge of the blade through the whole length and cut it.Now, expose and cut the decidui. Transfer them into a dish with growth medium. Dissect the decidui to extract the embryos.Transfer the embryos into a 60 millimeter Petri dish containing warm growth medium. With a pair of fine forceps, carefully cut the tail and the head of the embryo, and leave only the trunk of the body. Carefully remove as much tissue as possible from the trunk without damaging the heart or pulmonary and aorta arteries.Insert a new stretched out needle into the tip of the mouth pipette, and carefully aspirate and load 10 microliters of a prepared plasmid mix into the stretched out needle. Place a single heart into a clean 60 millimeter Petri dish containing prewarmed PBS. Place the dish under a dissecting microscope.Adjust the distance between the poles of positive and negative electrodes to about four millimeters. Next, use a needle in the mouth pipette to gently puncture the most superficial layer of the heart. Transfer one to two microliters of the plasmid mix into the heart.Remove the needle carefully. Hold the electrode in place so that the heart is located between both poles. Then, electroporate the heart.Transfer the electroporated heart to a new 12 well plate, containing one milliliter of growth medium. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide until analysis.

Extraktion och elektroporering av musembryohjärta som en transgenesmetod

Electroporated Mouse Cardiac Cell Isolation for Cell Sorting and Analysis

To begin, obtain electro operated mouse hearts extracted from wild type CD1 mice, 12 to 12.5 days after crossing. Transfer a heart into a tube containing one milliliter of ice cold digestion medium. With a syringe, apply gentle pressure on the heart a few times to chop it.Incubate the chopped tissue on a hot plate at 37 degrees Celsius for 45 minutes at 600 rotations per minute. Then filter the digestion mixture through a 70 micrometer cell strainer into a new tube. Next, filter the pass through volume again with a 40 micrometer cell strainer into a new 50 milliliter tube.Pipette 500 microliters of the FBS into the filtrate. Make up the volume up to 30 milliliters with cold DMEM. Centrifuge the suspension at 240G for 10 minutes at room temperature.Discard the supernatant. Then place the tube upside down on a paper towel to dry completely. For cell sorting, resuspend the cells in 300 microliters of sorting buffer.Finally, add 0.3 microliters of DAPI and perform cell sorting. The hearts were beating and appeared to be in good condition 24 hours post electroporation. The myocardium and epicardium did not show any signs of apoptosis.Analysis of the fluorescence activity showed a mosaic GFP signal in almost all four chambers of the heart.