Questo lavoro è stato sostenuto dal Ministero della Scienza e della Tecnologia (MOST 104-2311-B-001-034) e l&#39;Istituto di Biologia Cellulare e organismica, Academia Sinica, Taiwan.

1. Costruire tsMARCM-ready mosche Uso del Required transgeni 11, 13 <ol><li> Generare la versione originale di mosche tsMARCM-ready da transgeni che vengono effettuate da singoli mosche 11 (vedi tabella 1). Condurre programmi di incrocio standard di mosca genetiche, che sono stati descritti in precedenza, mettendo più transgeni nelle stesse scorte Fly 13. <ol><li> In una linea parentale, assemblare i transgeni di FRT 40A, UAS-mCD8 :: GFP (primo giornalista, mCD8 :: GFP, espressione del transgene è sotto il controllo del promotore upstream sequenza di attivazione, che produce un reporter membrana legato del gruppo murino di differenziazione 8 proteina fusa con la proteina fluorescente verde), e UAS-rCD2RNAi (il soppressore che inibisce l&#39;espressione del secondo giornalista, l&#39;RNAi contro l&#39;espressione di cluster di ratto of differenziazione 2, RCD2) sul braccio sinistro del cromosoma 2 nd. Posto conducente -GAL4 sulla X o 3 ° cromosoma, se possibile specifici tessuti. </li><li> In altra linea dei genitori, assemblare i transgeni di FRT 40A, UAS-RCD2 :: RFP (il secondo giornalista, RCD2 :: RFP, l&#39;altro giornalista membrana-tethered composto da RCD2 proteina fusa con la proteina fluorescente rossa), e UAS-GFPRNAi (il soppressore che inibisce l&#39;espressione di mCD8 :: GFP; RNAi contro l&#39;espressione di GFP) sul braccio sinistro del cromosoma 2 nd. Posizionare heat-shock (HS) -FLP o FLP tessuto-specific- sulla X o 3 ° cromosoma, se possibile. </li></ol></li><li> Generare la nuova versione di mosche tsMARCM-ready da transgeni che vengono effettuate da singoli mosche 14 (vedi tabella 1). Condurre mosca genetica schemi di incrocio standard, che sono stati descritti previously, mettendo più transgeni nelle stesse scorte Fly 13. <ol><li> In una linea parentale, assemblare i transgeni di un sito FRT ed UAS-mCD8.GFP.UAS-rCD2i (un transgene combinato di mCD8 :: GFP e il soppressore che inibisce l&#39;espressione di RCD2 :: RFP) insieme nello stesso braccio il 2 ° o 3 ° cromosoma (coppie appropriate di transgeni di FRT e UAS-mCD8.GFP.UAS-rCD2i vengono riportati in Tabella 1). Posizionare conducente tessuto-specifico GAL4 sui cromosomi diversi cromosoma del sito FRT prescelto, se possibile. </li><li> In altra linea parentale, assemblare i transgeni di un sito FRT ed UAS-rCD2.RFP.UAS-GFPi (l&#39;altra transgene combinato di RCD2 :: RFP e il soppressore che inibisce l&#39;espressione di mCD8 :: GFP) nello stesso braccio dei 2 ° o 3 ° cromosomiche (appropriate coppie di transgeni di FRT </em&gt; e UAS-rCD2.RFP.UAS-GFPi sono indicati nella Tabella 1). Inserire hs-FLP o tessuto-specifici FLP su cromosomi diversi cromosoma del sito FRT prescelto, se possibile. </li></ol></li></ol> 2. Croce tsMARCM pronto vola a generare cloni tsMARCM a loro progenie <ol><li> Mosche Croce tsMARCM-ready insieme (ad esempio, mettere 10-15 mosche maschio dal punto 1.1.1 o 1.2.1 e 20-30 femmina vergine vola dal punto 1.1.2 o 1.2.2 insieme) in un flaconcino fly-cibo con fresco pasta di lievito -made sulla parete del flaconcino. </li><li> Mantenere le incrociate mosche tsMARCM-ready a 25 ° C per 2 giorni per migliorare le possibilità di accoppiamento prima di raccogliere uova fecondate. </li><li> trasferire Spesso i incrociate mosche tsMARCM-ready in flaconcini appena yeasted per evitare affollamento (toccare le mosche o utilizzare anidride carbonica per anestetizzare le mosche per facilitare il trasferimento; tenere non più di 80-100 uova fecondate in 10 mL fly-alimentare tramiteL Per evitare troppi larve tratteggiata). </li><li> Culture gli animali tsMARCM (vale a dire, uova fecondate, un esempio del genotipo di animali tsMARCM, utilizzato in figura 3, è hs-FLP [22], w / w; UAS-mCD8 :: GFP, UAS-rCD2RNAi, FRT 40A , GAL4-GH146 / UAS-RCD2 :: RFP, UAS-GFPRNAi, FRT 40A; +; +) che sono state previste nelle fiale in serie trasferiti a 25 ° C fino a quando si sviluppano alle fasi desiderata (ad esempio, gli embrioni, larve, o pupe). </li><li> Posizionare le fiale trasferite che contengono animali tsMARCM allo stesso stadio di sviluppo in un bagno 37 ° C acqua per 10, 20, 30, 40, e 50 minuti per determinare il tempo ottimale di heat-shock che induce l&#39;espressione di FLP per generare la cloni tsMARCM di interesse. Ignorare questo passaggio se FLP tessuto-specific- viene utilizzato nell&#39;esperimento tsMARCM (hs-FLP è preferito per lignaggio neurale analisi, le ragioni di questa preferenza sono stati descritti in precedenza 15). NOTA: Ripetere il passaggio 2,5 utilizzando il tempo ottimale di heat-shock in esperimenti tsMARCM successivi se più tsMARCM cloni di interesse sono ricercati; in generale, più l&#39;espressione FLP è indotta in hs-FLP [22] rispetto al hs-FLP [1] con lo stesso tempo di calore-shock. </li><li> Cultura le calore scioccato animali tsMARCM a 25 ° C fino a quando non hanno raggiunto la fase di sviluppo appropriato, quindi passare al punto 3. </li></ol> 3. Preparare, Mordente, e Brains Monte Fly contenenti tsMARCM cloni <ol><li> Preparare piatti pinze-protezione. Miscelare la parte A (30 g) e la parte B (3 g) del Kit elastomero di silicone con carbone attivo (0,25 g). Versare il composto in piatti appositi. </li><li> Sezionare larvali, pupa, o cervello adulto fuori degli animali tsMARCM in 1x PBS (PBS) utilizzando una pinza su un piatto pinze protezione viste attraverso un microscopio dissezione. NOTA: Un protocollo per la dissezione mosca cervello è stato discribed in precedenza 16. </li><li> Fissare il cervello mosca in un posto piatto di vetro con 1x PBS contenente 4% di formaldeide a temperatura ambiente per 20 min. Elaborare i cervelli volano in questo posto piatto di vetro per il resto dei passaggi. </li><li> Risciacquare il cervello volare tre volte con 1% PBT (1x PBS contenente 1% Triton X-100) e lavarli tre volte per 30 minuti ciascuno in 1% PBT (risciacquo: rimuovere PBT e aggiungere nuovi PBT rapidamente; lavare: rimuovere PBT, aggiungere nuove PBT, e incubare il cervello mosca nel nuovo PBT utilizzando un agitatore orbitale). </li><li> Incubare le cervelli mosca con la miscela di anticorpi primari notte a 4 ° C o per 4 ore a temperatura ambiente. Un esempio della miscela di anticorpi primari è rat anticorpo anti-CD8 (1: 100), coniglio anticorpo anti-DsRed (1: 800), e Bruchpilot anti-anticorpo di topo (BPA) (1:50) in 1% PBT contenente 5% di siero normale di capra (NGS). </li><li> Risciacquare il cervello volare tre volte con 1% PBT, e poi lavarli tre volte per 30 minuti ciascuno con 1% PBT. </li><li&gt; Incubare cervello mosca con la miscela di anticorpi secondari notte a 4 ° C o per 4 ore a temperatura ambiente. Un esempio della miscela di anticorpi secondari è anticorpi IgG di capra anti-topo coniugato con colorante verde fluorescente (1: 800), anticorpi IgG anti-coniglio di capra coniugato con giallo colorante fluorescente (1: 800), e di capra anti-topo anticorpi IgG coniugato con lontano-rosso-colorante fluorescente (1: 800) in 1% PBT contenente il 5% NGS. </li><li> Risciacquare il cervello volare tre volte con 1% PBT, e poi lavarli tre volte per 30 minuti ciascuno con 1% PBT. montarli su un micro diapositiva con un reagente anti-tempra. Mettere un vetro di copertura micro sul micro vetrino per coprire e proteggere il cervello mosca. Sigillare i bordi del micro vetrino di copertura in vetro e micro con smalto trasparente. Memorizzare questi montati e sigillati volare campioni di cervello a 4 ° C. </li></ol> 4. Prendere, di processo, e analizzare le immagini fluorescenti di tsMARCM cloni <ol><li> Cattura immagini fluorescenti di cloni fr tsMARCMom i campioni creati nel passaggio 3,8 utilizzando il sistema di microscopia confocale di scelta. </li><li> Pile progetto di tsMARCM immagini fluorescenti confocale in due dimensioni, immagini appiattite utilizzando il software di elaborazione delle immagini fornito con il sistema di microscopia confocale di scelta (vedi Figura 3B - 3E, 3G - 3J, 3L - 3O, e 3Q - 3T per esempi di appiattito immagini fluorescenti confocale). </li><li> Contare il numero di cellule dei lati multi-cellulare-NB dei cloni tsMARCM utilizzando un software di elaborazione delle immagini appropriata (ad esempio, la funzione &quot;Cell Counter&quot; in ImageJ 17; vedi Figura 3E, 3J, 3O e 3T per gli esempi della cellula corpi dei neuroni). </li></ol>

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Gli autori dichiarano di non avere interessi finanziari concorrenti.

I passaggi critici all&#39;interno del protocollo I passi 1.1.1, 1.2.1, 2.3, 2.5 e 3.2 sono fondamentali per ottenere buoni risultati tsMARCM. Driver -GAL4 tessuto-specifica che non esprimono GAL4 in progenitori neurali sono preferiti per passi 1.1.1 e 1.2.1. Evitare di sovraffollamento gli animali cresciuti in fiale fly-alimentari nella fase 2.3. Poiché la latenza di induzione, il livello di espressione di FLP upon heat-shock, e il tempo medio di divisione dei progenitori neurali <em...

Il cervello, costituito da un vasto numero e diversi tipi di neuroni, dota gli animali con la capacità di percepire, di processo e rispondere alle sfide provenienti dal mondo esterno. I neuroni dell&#39;adulto Drosophila cervello centrale sono derivati da un numero limitato di cellule staminali neurali, chiamati neuroblasti (NBS), durante lo sviluppo 1, 2. La maggior parte di NBS partecipare a neurogenesi cerebrale in Drosophila sottoposti divisione asimmetrica per generare NBs auto-rinnovamento e cellule madri gangliari (GMC), e le GMCs poi passare attraverso un altro giro di divisione per la produzione di due cellule figlie che si differenziano in neuroni 3 (Figura 1A ). A causa della complessità della morfologia neuronale e le sfide connesse con l&#39;identificazione neuroni specifici, una tecnologia positiva etichettatura mosaico, analisi mosaico con un marker delle cellule reprimibile (MARCM), è stato inventato per consentire la visualizatione di un singolo neurone o un piccolo sottoinsieme di neuroni dalla popolazione di neuroni circostanti, senza etichetta 4. MARCM utilizza il sistema / target riconoscimento FLP (FRT) mediare ricombinazione sito-specifica tra cromosomi omologhi all&#39;interno di una cella di divisione precursore portando un allele eterozigote di un gene repressore, la cui espressione normalmente inibisce l&#39;espressione di un gene reporter 4 flippase (FLP). Dopo la divisione mitotica, i cromosomi ricombinanti sono segregati in cellule gemelle, tale che una cella contiene alleli omozigoti del gene repressore e l&#39;altra cella ha repressore gene-espressione del reporter in quella cella (ei suoi discendenti) non è più bloccato 4. Tre modelli clonali si trovano di solito in un esperimento MARCM quando FLP è stocasticamente indotta in NBs o GMCs: unicellulari e due celle-GMC cloni, che raffigurano la morfologia dei neuroni a resolutio cella singolan, e multi-cellulare-NB cloni, che rivelano interi modelli morfologici di neuroni derivati da una NB comune (Figura 1B). La tecnica MARCM è stato ampiamente applicato in Drosophila studi neurobiologici, compresa l&#39;identificazione di tipo neuronale per ricostruire le reti di cablaggio in tutto il cervello, neurali lignaggio analisi per rivelare la storia dello sviluppo dei neuroni, la caratterizzazione fenotipica delle funzioni dei geni coinvolti nella determinazione del fato cellulare, e la morfogenesi neuronale e differenziazione studia 5, 6, 7, 8, 9, 10. Poiché MARCM convenzionale etichetta solo una delle due cellule figlie (e linee) dopo l&#39;evento di ricombinazione mitotica indotta, informazioni potenzialmente utili dal lato marcato viene persa. Questa limitazione osta all&#39;applicazione della base MSistema ARCM a ad alta risoluzione le analisi di molti lignaggi neuronali che si attivano destino delle cellule in tempo veloce o per la precisione di analisi delle funzioni dei geni nei neuroni identici di diversi animali 11, 12. Twin-loco MARCM (tsMARCM) è un sistema avanzato che etichette neuroni derivati da un progenitore comune con due colori distinti, che permette il recupero delle informazioni utili da entrambi i lati delle celle gemelle, superando la limitazione del sistema MARCM originale 11 ( Figura 2A - 2C). Nel sistema tsMARCM, due interferenza RNA (RNAi) a base di soppressori sono situati trans siti di cromosomi omologhi all&#39;interno di una cellula precursore, e l&#39;espressione di tali soppressori inibiscono in maniera indipendente l&#39;espressione dei rispettivi reporter (Figura 2B). A seguito di site-specific ricombinazione mitotica mediata attraverso il sistema FLP / FRT, la TWsoppressori o basati RNAi diventare segregati in cellule gemelle per consentire l&#39;espressione del reporter distinti (Figura 2B). Due modelli clonali, cella singola associata con cloni unicellulari e due celle-GMC associati con cloni multi-cellulari-NB, sono in genere visti in un esperimento tsMARCM (Figura 2C). Informazioni derivato da un lato delle celle singoli possono essere utilizzati come riferimento per l&#39;altro lato, consentendo ad alta risoluzione analizza neurale lineage, come nascita-incontra i neuroni marcati, e fenotipica analisi dei neuroni identici in diversi animali per l&#39;indagine precisa della funzione del gene neurale 11, 12. Qui, vi presentiamo un protocollo passo-passo che descrive come condurre un esperimento tsMARCM, che può essere utilizzato da altri laboratori per ampliare gli studi di sviluppo neurale (nonché lo sviluppo di altri tessuti, se del caso) in Drosophila.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carbon dioxide (CO2)</td>
			<td>Local vendor</td>
			<td>n.a.</td>
			<td>Anesthetize flies</td>
		</tr>
		<tr>
			<td>CO2 pad</td>
			<td>Local vendor</td>
			<td>n.a.</td>
			<td>Sort, cross and transfer flies</td>
		</tr>
		<tr>
			<td>10x PBS pH 7.4</td>
			<td>Uniregion Bio-Tech (or other vendors)</td>
			<td>PBS001</td>
			<td>Dissect, rinse, wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>Fix fly brains</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Rinse, wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>Normal goat</td>
			<td rowspan="2">Jackson ImmunoResearch&#160;</td>
			<td rowspan="2">005-000-121</td>
			<td rowspan="2">Immunostain fly brains</td>
		</tr>
		<tr>
			<td>serum</td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD8 antibody</td>
			<td>Invitrogen</td>
			<td>MCD0800</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Rabbit anti-DsRed antibody</td>
			<td>Clonetech</td>
			<td>632496</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Mouse anti-Brp antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Goat anti-rat IgG antibody conjugated with Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11006</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG antibody conjugated with Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A11035</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG antibody conjugated with Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A21236</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>SlowFade gold antifade reagent</td>
			<td>Molecular Probes</td>
			<td>S36936</td>
			<td>Mount fly brains and protect quenching of fluorescence&#160;</td>
		</tr>
		<tr>
			<td>PYREX 9 depression glass spot plate</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td>Dissect, rinse, wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>World Precision Instruments</td>
			<td>SYLG184</td>
			<td>Make black Sylgard dishes to protect forceps during brain dissection</td>
		</tr>
		<tr>
			<td>Activated charcoal</td>
			<td>Sigma-Aldrich</td>
			<td>242276-250G</td>
			<td>Make black Sylgard dishes</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td>Dissect and mount fly brains</td>
		</tr>
		<tr>
			<td>Micro slide</td>
			<td>Corning</td>
			<td>2948-75x25</td>
			<td>Mount fly brains</td>
		</tr>
		<tr>
			<td>Micro cover glass No.1.5</td>
			<td>VWR International</td>
			<td>48366-205</td>
			<td>Mount fly brains</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Local vendor</td>
			<td>not available</td>
			<td>Seal micro cover glass on micro slides</td>
		</tr>
		<tr>
			<td rowspan="2">Incubator&#160;</td>
			<td>Kansin Instruments</td>
			<td rowspan="2">LTI603</td>
			<td rowspan="2">culture flies at 25&#176;C</td>
		</tr>
		<tr>
			<td>(or other vendors)</td>
		</tr>
		<tr>
			<td rowspan="2">Water bath</td>
			<td>Kansin Instruments</td>
			<td rowspan="2">WB212-B2</td>
			<td rowspan="2">Induce heat-shock in flies at 37&#176;C</td>
		</tr>
		<tr>
			<td>(or other vendors)</td>
		</tr>
		<tr>
			<td rowspan="2">Orbital shaker</td>
			<td>Kansin Instruments</td>
			<td rowspan="2">OS701</td>
			<td rowspan="2">Wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>(or other vendors)</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Leica</td>
			<td>EZ4</td>
			<td>Sort, cross and transfer flies; Dissect, rinse, wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss (or other vendors)</td>
			<td>LSM 700 (or other models)</td>
			<td>Image tsMARCM clones</td>
		</tr>
		<tr>
			<td>image-processing software 1 
			(e.g., Zeiss LSM image browser)&#160;</td>
			<td>Zeiss</td>
			<td>not available</td>
			<td>Project stacks of confocal images</td>
		</tr>
		<tr>
			<td>image-processing software 2 
			(e.g., Image-J)&#160;</td>
			<td>not available</td>
			<td>not available</td>
			<td>Count cell number of tsMARCM clones</td>
		</tr>
	</tbody>
</table>

cell lineage analyses and gene function studies using twin-spot marcm

M osaic un nalisi con una r epressible c ell m Arker (MARCM) è un sistema di etichettatura mosaico positivo che è stato ampiamente applicato in Drosophila studi neurobiologici per descrivere morfologie intricate e di manipolare la funzione dei geni in sottoinsiemi di neuroni all&#39;interno altrimenti non marcato e imperturbabile organismi. mosaici genetici generati nel sistema MARCM sono mediati attraverso la ricombinazione sito-specifica tra cromosomi omologhi all&#39;interno di divisione delle cellule precursori per la produzione di entrambi marcati (cloni marcm) e cellule figlie non marcate durante la mitosi. Una estensione del metodo MARCM, chiamato twin-spot MARCM (tsMARCM), etichette sia delle cellule gemelle derivate da un progenitore comune con due colori distinti. Questa tecnica è stata sviluppata per consentire il reperimento di informazioni utili da entrambe le emi-linee. Con completo Analizzando diverse coppie di cloni tsMARCM, il sistema consente di tsMARCM ad alta risoluzione neurale lignaggio Mapping di rivelare l&#39;esatto nascita ordine dei neuroni marcati prodotte dalle cellule progenitrici comuni. Inoltre, il sistema tsMARCM estende anche studi di funzione genica consentendo l&#39;analisi fenotipica dei neuroni identici di diversi animali. Qui, descriviamo come applicare il sistema tsMARCM per facilitare gli studi di sviluppo neurale in Drosophila.

Il sistema tsMARCM è stato utilizzato per facilitare neurale lignaggio analisi e studi la funzione del gene per il recupero delle informazioni importanti sui neuroni derivati ​​da NBs comuni. Il sistema è stato utilizzato per identificare la maggior parte (se non tutti) i tipi neuronali, determinare il numero di cellule di ogni tipo neuronale, e accertare la nascita di ordine di questi neuroni 11, 12, <sup class=...

Watch this Scientific Journal Video about Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM at JoVE.com

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Qui, presentiamo un protocollo per una tecnica di etichettatura mosaico che permette la visualizzazione dei neuroni derivati ​​da una cellula progenitrice comune in due colori distinti. Questo facilita l&#39;analisi lignaggio neurale con la capacità di singoli neuroni nascita aggiornamento e la funzione del gene studiando negli stessi neuroni di diversi individui.

Le analisi delle cellule lignaggio e studi la funzione del gene Impiegando doppi loco MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

cell-lineage-analyses-and-gene-function-studies-using-twin-spot-marcm

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.9K Views.  Academia Sinica.  Qui, presentiamo un protocollo per una tecnica di etichettatura mosaico che permette la visualizzazione dei neuroni derivati ​​da una cellula progenitrice comune in due colori distinti. Questo facilita l'analisi lignaggio neurale con la capacità di singoli neuroni nascita aggiornamento e la funzione del gene studiando negli stessi neuroni di diversi individui. 

Video: Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

Trasferimento efficiente Gene in Chick retine per cellulari primarie Cultura Studies: An<em&gt; Ex-ovo</em&gt; Elettroporazione Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Ricerca

Biologia dello sviluppo

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Colture organotipiche Slice di studi di post-natale neurogenesi

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Un metodo per Lineage Tracing di cellule corneali Utilizzando multicolori topi reporter fluorescente

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

Co-localizzazione di marcatori di cellule lignaggio e il segnale di pomodoro

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Manipolazione funzionale delle materne genica Utilizzando<em&gt; In Vitro</em&gt; La maturazione degli ovociti in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Studi struttura-funzione nel topo cellule staminali embrionali Uso ricombinasi mediata Cassette Scambio

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR-mediata di perdita di funzione di analisi in granuli cerebellari cellule utilizzando nell'Utero basati su elettroporazione trasferimento del Gene

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

L'analisi di espressione genica tramite rilevazione di attività di β-galattosidasi in embrioni di topo intero

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Analisi trascrittomica unicellulare delle cellule Endocrine del pancreas del Mouse

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Le analisi delle cellule lignaggio e studi la funzione del gene Impiegando doppi loco MARCM

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Trasferimento efficiente Gene in Chick retine per cellulari primarie Cultura Studies: An