Gli autori desiderano ringraziare Jane Johnson per il Math1, Ngn1, e gli anticorpi Ptf1a e Connie Cepko per la pCAG :: GFP plasmide. Questo lavoro è stato finanziato dal NIH 1R15HD059922 R15-01.

1. La preparazione prima Elettroporazione <ol><li> Amplificare il DNA per l&#39;elettroporazione di un maxi prep (Prime-It or Qiagen). La concentrazione del DNA dovrebbe essere un minimo di 1 mg / ml per l&#39;assorbimento efficiente. </li><li> Rimuovere 495 pl di DNA e mescolare con 5 pl di 0,01% verde veloce in 1 x PBS (tampone fosfato salino) in una provetta da microcentrifuga. </li></ol> 2. Harvest embrionali <ol><li> Stabilire accoppiamenti cronometrati di topi CD-1 (Harlan). Verificare la presenza di tappi vaginali e considerano la data si osserva un tappo vaginale come il giorno embrionale (E) 0.5. Gli embrioni saranno raccolte 11 giorni dopo la visualizzazione spina (E11.5). </li><li> Posizionare strumenti sterilizzati in etanolo 70%. Trattare una cappa a flusso laminare con luce UV per almeno un&#39;ora prima dell&#39;uso. Spray hood, vassoio di dissezione, e dissezione portata con il 70% di etanolo. Pre-calore 1 X PBS a 37 ° C. </li><li> Sul E11.5 l&#39;eutanasia della femmina in base alle condizioni approvate dal InstitutComitato ionale per la cura e l&#39;uso degli animali (ICCUA). Mettere nel cassetto dissezione cappa a flusso laminare. Spruzzare verso il basso l&#39;addome della femmina con il 70% di etanolo. </li><li> Aprire la cavità peritoneale e appuntare le pareti al vassoio dissezione. Estrarre le corna uterine in modo che appoggi all&#39;interno della cavità peritoneale. </li><li> Tagliare con cautela aprire la parete della tromba uterina. Utilizzare un cucchiaio 20 mm (Belle Strumenti Scientifici) per rimuovere delicatamente l&#39;embrione nel sacco vitellino lontano dalla placenta. Embrioni luogo in un piatto di coltura tissutale 100 millimetri pre-riempite con 10 ml di PBS sterile 1 X che è stato preriscaldato in 2.2. </li><li> Ripetere l&#39;operazione per tutti gli embrioni presenti. </li></ol> 3. Elettroporazione di embrioni E11.5 (figura 1) <ol><li> Utilizzo di un cucchiaio 20 mm, trasferire il primo embrione in un piatto 100 millimetri preriempita con 10 ml di 1XPBS sterili che è stato preriscaldato a 37 ° C. </li><li> Utilizzare pinze cm 11 x 0,05 con una punta di 0,02 mm (Belle Strumenti Scientifici) di ri attenzionespostare ed eliminare il sacco vitellino. </li><li> Embrione di posizione in modo che il suo lato dorsale è rivolto verso l&#39;alto in modo che assomigli il cartone in Figura 1A. Utilizzare 7 pale elettrodi mm (Harvard Apparatus) a tenere delicatamente l&#39;embrione. Gli elettrodi devono essere posizionati su entrambi i lati del tubo neurale a livello del ventricolo rombencefalo quarto. Il ventricolo è visibile ad occhio nudo, ma utilizzando un microscopio di dissezione può facilitare il posizionamento accurato paddle. Posizionamento delle palette è fondamentale per determinare la regione che riceve il DNA elettroporata. Se il labbro inferiore romboidale (LRL) del rombencefalo dorsale embrionale si desidera, pale deve essere posto in modo tale che essi sono direttamente fiancheggia la parte più larga dell&#39;apertura quarto ventricolo. </li><li> Utilizzare una siringa da 1 cc a redigere il DNA plasmidico miscelato con 0,01% Verde Fast. 500 pl della miscela deve essere sufficiente per l&#39;elettroporazione di almeno 8-10 embrioni (cfr. 3.5). </li><li> Delicatamente perforare la ov roofplateerlying quarto ventricolo con un ago 25G tubercolina 5/8 attaccato alla siringa 1 cc e iniettare il DNA-colorante miscela nel ventricolo. Iniezioni successo sono caratterizzate dal DNA-colorante miscela riempimento dell&#39;intero sistema ventricolare (Figura 1C). La quantità di DNA-colorante miscela tipicamente iniettato è inferiore a 50 pl. Importi esatti sono variabili tra embrioni come una porzione della miscela tende a fuoriuscire dal ventricolo nel PBS che circonda l&#39;embrione. Un mezzo alternativo di consegna del DNA-dye miscela sarebbe attraverso l&#39;accesso al sistema ventricolare perforando il velo che ricopre il mesencefalo. Ancora, iniezioni di successo sono caratterizzati dal DNA-colorante miscela riempimento dell&#39;intero sistema ventricolare. </li><li> Consegnare cinque impulsi quadrati utilizzando un generatore di impulso elettrico (BTX) e 7 pale elettrodi mm. Ogni impulso è 50 V della durata di 5 ms per impulso con 500 ms tra ogni impulso. Il tessuto più vicino all&#39;elettrodo caricata positivamente quindi take il plasmide. </li></ol> 4. Cultura di embrioni <ol><li> In una cappa a flusso laminare, riempire i pozzetti esterne di un 12-ben piatto di coltura con 2 mL di DMEM/F12 mezzo supplementato con 10% siero fetale bovino, 5% siero equino, 1% glutammina, 1% di penicillina / streptomicina che è stato preriscaldato a 37 ° C. Le condizioni di coltura sono stati adattati da de Diego e colleghi 15. </li><li> In embrioni cappa a flusso laminare pizzico di midsection (sotto il cuore) con una pinza e rimuovere parte posteriore dell&#39;embrione. Posizionare la porzione anteriore in uno dei pozzetti riempiti del 12-ben piatto di coltura. </li><li> Ripetere l&#39;operazione per tutti gli embrioni. Riempire solo i pozzi esterni del 12 pozzetti per evitare la contaminazione delle culture. </li><li> Cultura embrioni in un incubatore a 37 ° C al 5% di CO 2. Espressione del plasmide elettroporate dovrebbe essere osservabile entro 24 ore. </li><li> In caso di tempi più lunghi la cultura essere desiderata, riempire i pozzetti su un nuovo piatto ben 12 con 2 ml dii supporti utilizzati in 4.1. Con un cucchiaio sterilizzato trasferire gli embrioni a un pozzo nella nuova piastra. Mettere di nuovo nel 37 ° C incubatore. La cultura per un massimo di 48 ore è possibile. </li><li> Quando il tempo di coltura desiderato viene raggiunto, fissare embrioni per analisi (vedi sotto). </li></ol> 5. Preparazione di embrioni per l&#39;analisi <ol><li> Risciacquare embrioni in 1 x PBS a 4 ° C per cinque minuti. Ripeti. </li><li> Fix embrioni per analisi in 2% paraformaldeide (PFA) in 1XPBS per 2 ore a 4 ° C. </li><li> Risciacquare embrioni in 1 x PBS a 4 ° C per cinque minuti. Ripeti. </li><li> Equilibrare embrioni in 30% saccarosio in 1 x PBS notte a 4 ° C. </li><li> Incorpora embrioni in temperatura ottimale di taglio (OCT) composto con un ghiaccio secco / etanolo bagno. Gli embrioni possono essere conservati a -20 ° C. </li><li> Embrioni sezione in un criostato (Leica), in 30 sezioni micron e montare su vetrini (VWR, Superfrost Plus). Conservare a -20 ° C. </li></ol> 6. ImmuAnalisi nohistochemistry <ol><li> L&#39;immunoistochimica è stata eseguita come descritto in 16. Diluizioni degli anticorpi primari utilizzati per questo studio includono coniglio α-GFP (Invitrogen) 1:2500; del mouse α-Mash1 (BD Biosciences) 1:100; coniglio α-Ngn1 (Jane Johnson) 1:5000; coniglio α-Ptf1a (Jane Johnson ) 1:2500; coniglio Math1 (Jane Johnson) 1:100. Incubare guida piatta, lato campione su un vassoio di colorazione a 4 ° C per una notte. </li><li> I vetrini sono stati analizzati su un microscopio composto (Olympus BX51). </li></ol> 7. Risultati rappresentativi Uno schema in figura 1A mostra l&#39;esperimento elettroporazione. Figura 1B mostra una vista sagittale di un embrione E11.5 prima della manipolazione. L&#39;embrione stesso dopo l&#39;iniezione del CAG plasmide contenente :: GFP in 0,01% verde rapido viene mostrato nella Figura 1C e rappresentante un embrione non fissata espositiva GFP unilateraleespressione nella dorsale rombencefalo 24 ore cultura seguente è mostrato nella figura 1D. L&#39;estensione della zona del LRL che viene correttamente elettroporata è variabile e sembra essere fortemente dipendente posizionamento degli elettrodi. Nei nostri studi è stato trovato che 52 su 65 (80%) degli embrioni elettroporate espressi con successo GFP. Tessuto è stato considerato essere correttamente elettroporata se era positivo per GFP nelle regioni localizzate su varie sezioni successivo fissaggio e l&#39;analisi immunoistochimica (vedi sotto). Gli embrioni che non hanno rispettato questi criteri sono stati segnati come tentativi falliti di elettroporazione. Ulteriore valutazione dell&#39;efficienza elettroporazione può essere accertata eseguendo immunoistochimica contro GFP su sezioni trasversali di embrioni elettroporate a livello del quarto ventricolo. Figura 2 mostra rappresentativi sezioni seriali da un embrione visualizzazione unilateraleEspressione di GFP. Figura 2A mostra uno schema idealizzato che illustra che il lato sinistro dell&#39;embrione era verso l&#39;elettrodo positivo. Sezioni trasversali (a livelli rappresentati dalla metà cartone di figura 2A) rivelare l&#39;espressione localizzata GFP esclusivamente sul lato sinistro del tessuto rombencefalo (figure 2C e 2D). L&#39;area elettroporata del LRL non estendere l&#39;intero asse anteriore-posteriore del tubo neurale come esame di sezioni 300 um rostrali o superiore a quella mostrata in Figura 2C non esprime GFP (Figura 2B). L&#39;utilità di questo test di manipolazione genica dipende dalla stabilità dei domini di espressione per le proteine ​​endogene. Il LRL è stato caratterizzato come in possesso di domini unico progenitore caratterizzate dalla espressione differenziale di fattori di trascrizione proneural (recensione in 1).Un sottoinsieme di questi fattori (Mash1, Math1, Ngn1 e Ptf1a) sono stati scelti per l&#39;analisi a causa della loro proposta e / o ruoli caratterizzati nel disciplinare di precerebellar sottotipi neurali nel LRL, oggetto di studi futuri 16-18. Tutte e quattro le proteine ​​hanno domini di espressione altamente caratteristici della rombencefalo caudale a E11.5 16-18. Abbiamo osservato che gli embrioni che sono stati collocati culture riuscito ad aumentare di dimensioni e inoltre non riesce a iniziare la produzione di epitelio plesso coroideo e invaginazione delle LRL e roofplate, eventi morfologici che si verificano tra E11.5 e E12.5 5. Sulla base di queste osservazioni è stato stabilito che il normale sviluppo di questi embrioni è stata interrotta o gravemente ritardata e il controllo comparabile per gli embrioni devono essere colte incolte embrioni a E11.5. Per garantire che la cultura ed elettroporazione non disturbare i livelli di proteine ​​endogene, abbiamo analizzato quattro diverse proteine ​​da immunohistochemistry (IHC) in 34 embrioni differenti che sono state elettroporate e quindi coltivate per almeno 24 ore. Tabella I mostra il numero di embrioni analizzati per ciascun marcatore e la percentuale degli embrioni analizzato che mantiene livelli normali proteine. Figura 3 mostra i dati rappresentativi IHC da due delle proteine ​​analizzati, Mash1 (Figure 3A e 3B) e Math1 (figure 3C e 3D). Abbiamo osservato che la maggior parte degli embrioni mantiene livelli normali di elettroporazione seguente espressione e cultura (figure 3b e 3d) rispetto agli embrioni di controllo a E11.5 (Figure 3A e 3C). È importante sottolineare che i domini di espressione caratteristici di queste proteine ​​non sono stati perturbato. <img src="/files/ftp_upload/3983/3983fig1.jpg" alt="Figura 1"/> Figura 1. Elettroporazione di embrioni a E11.5. (A) Schema di The elettroporazione esperimento. Un embrione E11.5 è isolato e il plasmide di espressione in 0,01% verde rapido viene iniettato nel quarto ventricolo. L&#39;embrione viene affiancata da pale elettrodi e sottoposto ad un impulso V 50 prima di essere messi in coltura (B) sagittale di un embrione E11.5 prima dell&#39;iniezione. (C) L&#39;embrione stesso E11.5 dopo l&#39;iniezione del plasmide di 0,01% verde Fast. (D) espressione rombencefalo unilaterale della GFP osservato in embrioni E11.5 dopo 24 ore di cultura. mb-mesencefalo; 4V quarto ventricolo. <img src="/files/ftp_upload/3983/3983fig2.jpg" alt="Figura 2"/> Figura 2. L&#39;espressione di GFP in Tissue elettroporata. (A) Il cartone sulla sinistra rappresenta il posizionamento degli elettrodi attorno a un embrione E11.5. Cartone mediana mostra l&#39;assorbimento e l&#39;espressione di GFP plasmide codificante sul lato sinistro dell&#39;embrione. Cartoon sulla destra è schematica di una sezione trasversale idealizzata presa attraverso l&#39;embrione in levels contrassegnata da linee nere in mezzo fumetto. (B-D) per la GFP immunoistochimica su sezioni trasversali attraverso un embrione elettroporate E11.5 dopo 24 ore di cultura. Le frecce indicano la (rp) roofplate che intrappola l&#39;anticorpo secondario. Le immagini sono prese a ingrandimento 10X. I livelli relativi delle sezioni mostrate sono rappresentati dalle linee orizzontali attraverso il cartone centrale in (A). mb-mesencefalo; l 4V quarto ventricolo; rp-roofplate. <img src="/files/ftp_upload/3983/3983fig3.jpg" alt="Figura 3"/> Figura 3. Espressione di proteine ​​endogene nel labbro inferiore rombico immunoistochimica per Mash1 (A e B) o Math1 (C e D) confrontare sezioni trasversali di embrioni E11.5 che non erano coltivate (A, C) con embrioni sono state elettroporate con CAG.: : GFP e coltivate per 24 ore (B e D). Immagini prese con ingrandimento 10X. <table border="1"><tbody><tr><td> Proneural Transcriptisu fattori analizzati </td><td> Numero di embrioni analizzati </td><td> Percentuale ritenuta Patterns normale espressione </td></tr><tr><td> Math1 </td><td> 15 </td><td> 86,7% </td></tr><tr><td> Mash1 </td><td> 12 </td><td> 83,3% </td></tr><tr><td> Ngn1 </td><td> 7 </td><td> 71,4% </td></tr><tr><td> Ptf1a </td><td> 6 </td><td> 100% </td></tr></tbody></table> Tabella I. Percentuale di embrioni elettroporate e colto di contenimento normali Proneural Domini fattore di trascrizione nel LRL.

<ol>
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La tecnica di elettroporazione in vitro presentato in questo studio è una nuova metodologia che può essere efficacemente utilizzata per manipolare l&#39;espressione genica in embrioni di età inferiore ai 12 giorni di gestazione. Posizionamento degli embrioni in cultura permette l&#39;espressione del gene introdotto e aggira la letalità osservata quando embrioni elettroporate possono rimanere in vivo. Questa tecnica permette la manipolazione di espressione genica in progenitori embrionali precedente...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Criostato </td><td> Leica </td><td> CM-1850 </td><td></td></tr><tr><td> Biologie punta Dumoxel trattati DUMONT forcipe </td><td> Belle Strumenti Scientifici </td><td> 11252-30 </td><td></td></tr><tr><td> 20 millimetri MORIA cucchiaio forato </td><td> Belle Strumenti Scientifici </td><td> 10370-17 </td><td></td></tr><tr><td> ECM 830 Onda quadra Generator Elettroporazione </td><td> BTX (VWR) </td><td> 47745-928 </td><td></td></tr><tr><td> Harvard Apparatus Tweezertrodes 7 mm * Elettrodi </td><td> BTX (Fisher) </td><td> BTX450165 </td><td></td></tr><tr><td> Fisher Isotemp CO 2 Incubator </td><td> Pescatore </td><td> 1325525 </td><td></td></tr><tr><td> NAPCO CO 2 Gas Regulator </td><td> Pescatore </td><td> 15497020 </td><td></td></tr><tr><td> 12 Bene piastre di coltura dei tessuti </td><td> BD Falcon (Fisher) </td><td> 877229 </td><td></td></tr><tr><td> HyClone Liquid media DMEM/F-12 (1:1), con L-glutammina e HEPES; 500mL </td><td> Thermo Scientific (Fisher) </td><td> SH3002301 </td><td></td></tr><tr><td> HyClone * Donor siero equino </td><td> Thermo Scientific (Fisher) </td><td> SH3007402 </td><td></td></tr><tr><td> Siero fetale bovino, qualificato, inattivato al calore </td><td> Invitrogen </td><td> 16140-063 </td><td></td></tr><tr><td> Cellgro * 10.000 UI di penicillina, 10.000 mg / ml di streptomicina </td><td> Mediatech (Fisher) </td><td> MT-30-002-CI </td><td></td></tr><tr><td> HyClone * L-Glutamina L-Glutammina; 200 mm in NaCl 0,85% </td><td> Thermo Scientific (Fisher) </td><td> SH3003401 </td><td></td></tr><tr><td> Fast-verde </td><td> Pescatore </td><td> AC41053-0250 </td><td> 0,01% </td></tr></tbody></table>

in vitro electroporation of the lower rhombic lip of midgestation mouse embryos

Il labbro rombico è uno neuroepitelio embrionale situato nel rombencefalo alla giunzione tra il tubo neurale e la roofplate del quarto ventricolo (recensione in 1). Il labbro rombico può essere suddiviso nel labbro superiore romboidale (URL) che comprende rhombomere 1 (R1) e genera neuroni del cervelletto e il labbro inferiore romboidale (LRL) che dà luogo a diversi lignaggi tronco neuronali 2-4. LRL derivati ​​comprendono i neuroni uditivi dei nuclei cocleari e quelle dei nuclei precerebellar che sono coinvolti nella regolazione dell&#39;equilibrio e il controllo motorio 5-8. Neurogenesi dalla LRL si verifica su una grande finestra temporale che abbraccia giorni embrionali (E) 9,5-16,5 5, 9. Differenti linee neuronali emergono dal LRL come cellule postmitotico (o sono nati) durante il giorno di sviluppo distinti durante questa finestra neurogena. Elettroporazione di costrutti di espressione genica può essere utilizzato permanipolare l&#39;espressione genica in progenitori LRL e potenzialmente in grado di cambiare il destino dei neuroni ottenuti da questa regione 10-12. Alterare l&#39;espressione genica dei progenitori LRL nel topo mediante elettroporazione in utero ha avuto molto successo per la manipolazione di lignaggi nati il giorno embrionale E12.5 o poi 10, 12-14. In elettroporazioni utero prima E12.5 non hanno avuto successo dovuto principalmente al mortalità associato con la puntura del roofplate quarto ventricolo, un passo necessario nella realizzazione di DNA esogeno che viene elettroporate nel LRL. Tuttavia, molti lignaggi LRL derivati ​​nascono dalla LRL prima di E12.5 9. Questi lignaggi anteriore nati includono i neuroni che compongono il reticolare laterale, cuneate esterna e inferiore nuclei olivare del sistema precerebellar che funzionano per collegare gli ingressi dal midollo spinale e corteccia al cervelletto 5. Per manipolare espressione nel LRLdi embrioni con meno di E12.5, abbiamo sviluppato un sistema in vitro in cui si trovano gli embrioni nella cultura dopo l&#39;elettroporazione. Questo studio presenta un metodo efficiente ed efficace per manipolare l&#39;espressione genica di progenitori LRL a E11.5. Gli embrioni elettroporate con la proteina fluorescente verde (GFP) guidato dal promotore CAG ampiamente attiva riproducibile espresso GFP dopo 24 ore di coltura. Un aspetto critico di questo saggio è che l&#39;espressione genica è alterata solo per l&#39;espressione del gene esogeno e non a causa di effetti secondari che derivano dalla elettroporazione e tecniche di coltura. E &#39;stato stabilito che i pattern di espressione dei geni endogeni restano indisturbati negli embrioni elettroporate e colta. Questo test può essere utilizzato per modificare il destino delle cellule che emergono dalla LRL di embrioni di età inferiore ai E12.5 attraverso l&#39;introduzione di plasmidi per la sovraespressione o abbattere (tramite RNAi) di diverse pro-fattori di trascrizione neurali.

Watch this Scientific Journal Video about In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos at JoVE.com

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Questo studio descrive lo sviluppo di uno<em&gt; In vitro</emElettroporazione&gt; tecnica che permette la manipolazione di espressione genica nel labbro inferiore rombica di embrioni midgestation.

In Elettroporazione vitro del labbro inferiore Rombico di embrioni di topo Midgestation

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

in-vitro-electroporation-lower-rhombic-lip-midgestation-mouse

Research

JoVE Journal

Neuroscience

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

10.3K Views.  University of Illinois at Springfield. Questo studio descrive lo sviluppo di uno In vitro tecnica che permette la manipolazione di espressione genica nel labbro inferiore rombica di embrioni midgestation.

Video: In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Ricerca

Neuroscienze

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Quantificare le attività di Cis-Regolamentazione Elementi nella retina di topo Elettroporazione Espianto

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Mouse in Utero elettroporazione: controllato Transefection Gene spazio-temporale

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Gene Transfer per l&#39;orecchio del mouse Sviluppo Inner<em&gt; In Vivo</em&gt; Elettroporazione

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell&#39;ippocampo mouse utilizzando<em&gt; In utero</em&gt; Elettroporazione

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Elettroporazione del romboencefalo tracciare traiettorie assonale e Obiettivi Synaptic in embrione di pollo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

La manipolazione genetica del topo sviluppo dell&#39;ipotalamo attraverso<em&gt; In utero</em&gt; Elettroporazione

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Un<em&gt; In-vitro</em&gt; Preparazione dei neuroni enterici isolati e glia dal plesso mienterico del topo adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.