Gli autori ringraziano il personale del nucleo Morfologia e Imaging, del nucleo Genetica Molecolare (Dipartimento di Oftalmologia e Scienze Visive) e del mouse Genetica Nucleo presso la Washington University di St. Louis School of Medicine. Gli autori sono supportati dal piccolo programma di sovvenzioni dal Centro McDonnell per Cellulare e Neurobiologia Molecolare, il Centro Speranza per disordini neurologici, il National Eye Institute (# R01EY022632 a DH), un Istituto Nazionale di Eye Centro Nucleo Grant (# P30EY002687) e un sovvenzione illimitata di ricerca per prevenire la cecità al Dipartimento di Oftalmologia e Scienze Visive.

Etica Dichiarazione: procedure che coinvolgono soggetti animali sono stati approvati dalla cura e l&#39;uso Comitato istituzionale animali (IACUC) presso la Washington University di St. Louis. 1. Allevamento Mouse e genotipizzazione <ol><li> Breed Tg (CAG-LacZ / EGFP-KASH2) topi con Tg (PCP2-Cre) per produrre topi Tg (PCP2Cre CAG-EGFP / KASH2) 19,20. Nota: Mentre il resto di questo protocollo concentra sull&#39;uso della linea Tg (PCP2-Cre) topo per restringere espressione EGFP-KASH2 alle cellule di Purkinje nel cervelletto, una procedura simile può essere seguita con qualsiasi altra Tg (Cre) mouse ( Figura 2A). </li><li> Eseguire genotipizzazione per individuare i cuccioli che ospitano sia transgeni EGFP e Cre come descritto in precedenza 19. </li></ol> 2. Preparazione dei Materiali per la dissezione e tessuto Collection <ol><li> Preparare 100 ml di 30% di saccarosio in PBS (30 g di saccarosio / 100 ml di PBS 1x) e conservare a -20 ° C. Il giorno del tessuto harvesting, disgelo e carico ~ 30-35 ml di soluzione di saccarosio in una siringa da 35 ml per perfusioni transcardial. </li><li> Preparare 40 ml di 4% PFA in PBS (10 ml 16% paraformaldeide e 30 ml PBS). Carico ~ 30-35 ml di 4% PFA in una siringa da 35 ml per perfusioni transcardial. </li><li> Riempire una piastra di coltura tissutale 10 centimetri con 10 ml di PBS per la dissezione dei tessuti. </li><li> Preparare un cocktail di ketamina / xilazina costituito da 16 mg / ml di ketamina e 3 mg / ml xilazina. </li><li> Preparare una soluzione di etanolo al 70% per la dissezione. </li></ol> 3. Dissection e tessuto Collection <ol><li> Usando una siringa da 1 ml, 500 ml di iniettare il cocktail ketamina / xylazina per 25 grammi di peso corporeo ed attendere ~ 15 min. In alternativa, utilizzare un altro metodo di sedazione istituzionalmente riconosciuto che è appropriato per il sistema biologico di interesse. </li><li> Su sedazione, pizzicare delicatamente la zampa posteriore per confermare la mancanza di risposta al dolore, riporre tutte le zampe su un vassoio di polistirolo con il lato dorsale delil mouse verso il basso. </li><li> Spruzzare la addome con 70% etanolo e fare un&#39;incisione dal basso addome alla sommità della gabbia toracica per accedere al cuore. </li><li> Effettuare una piccola incisione nell&#39;atrio destro del cuore e inserire la siringa contenente la soluzione di saccarosio al 30% nel ventricolo sinistro del cuore. </li><li> Profumato la soluzione ad una velocità di ~ 2-5 ml per min. Quando è vuoto, rimuovere la siringa e ripetere con la soluzione PFA 4%. </li><li> Sblocca il mouse dal vassoio di polistirolo e decapitarlo per accedere al cervello. </li><li> Togliere la pelle intorno alla testa dell&#39;animale con pinze e forbici. Fare un buco nella parte posteriore del cranio con un ago e rimuovere con attenzione il cranio con forbici e pinze dissezione. </li><li> Una volta che il cervello è chiaramente accessibile, utilizzare pinze per rimuovere il cervelletto e metterlo in PBS. Bisect cervelletto nel piano sagittale con un bisturi e trasferire la soluzione di saccarosio al 30% per una notte a 4 ° C. Nota: Per prepararesono sezioni di paraffina, fissano il cervelletto notte a 4 ° C in PFA 4% prima di incorporamento dei tessuti e sezionamento. </li></ol> 4. Tissue Processing e sezionamento <ol><li> Preparare una sospensione di ghiaccio secco e butano 2-metil. Consentire la temperatura del bagno si stabilizzi per 5 min. </li><li> Riempire un cryomold con OCT e mettere la metà del cervelletto bipartita nello stampo. </li><li> Utilizzando un microscopio da dissezione e una coppia di pinze posizione cervelletto modo che la superficie bisecata (vicino alla linea mediana del cervelletto) è rivolto verso l&#39;alto. </li><li> Trasferire il cryomold all&#39;impasto ghiaccio secco per 5 minuti fino a quando il composto ottobre ha formato una solida matrice bianco intorno il campione di tessuto. Nota: I campioni congelati possono essere conservati a -20 ° C, se necessario. </li><li> Etichetta pretrattate diapositive poli-lisina per la raccolta sezioni di tessuto e preparare il criostato secondo le istruzioni del produttore per raccogliere 15 micron sezioni del Cerebellum. Per raccogliere sezioni contattare attentamente la sezione PTOM il lato rivestito poli-lisina della slitta. Per mantenere l&#39;orientamento della sezione, non ruotare la slitta durante questa fase. </li><li> Sezioni di tessuto Conservare a -20 ° C fino a procedere con la colorazione e l&#39;imaging. </li></ol> 5. La colorazione e imaging di Cervelletto Sezioni <ol><li> Preparare le seguenti soluzioni prima della colorazione dei tessuti: ~ 50 ml di PBS ~ 5-10 ml di PFA 4% in PBS ~ 10-20 ml di Triton X-100 0,5% siero asino, 10% in PBS </li><li> Sezioni cerebellari disgelo. È importante sottolineare che ridurre al minimo la loro esposizione a qualsiasi fonte di luce continua e intensa per evitare sbiadimento di EGFP. </li><li> Con una penna barriera idrofobica, disegnare un rettangolo intorno alle sezioni di tessuto. </li><li> Post-fissare le sezioni con 4% PFA per 5 min. Usi abbastanza liquido da coprire l&#39;intera area delimitata dalla barriera idrofoba (di solito ~ 500 microlitri). Utilizzare un aspirapolvere o pipetta, rispostare il fissativo essere particolarmente attenti a non disturbare i sezioni di tessuto. </li><li> Con una pipetta, applicare delicatamente PBS sul vetrino senza disturbare le sezioni di tessuto. Dopo 5 min utilizzare il vuoto o pipetta per rimuovere con attenzione il liquido. Ripetere la PBS sciacquare un totale di tre volte. </li><li> Usando una pipetta, applicare con cura la diluizione appropriata di anticorpi primari (3 ml calbindin e / o anticorpi Nesprin2 per 900 ml di soluzione di) nello 0,5% Triton X-100, il 10% di siero asino in PBS. Incubare per un&#39;ora a temperatura ambiente al buio. Nota: EGFP-KASH2 sarà visibile mediante microscopia a fluorescenza diretta su sezioni congelate, e non è necessario utilizzare un anticorpo anti-EGFP. Tuttavia, se sono stati preparati sezioni di paraffina, l&#39;imaging di EGFP-KASH2 richiede immunolocalizzazione con un anticorpo anti-EGFP. </li><li> Rimuovere la soluzione di anticorpi e risciacquare con PBS per tre volte. </li><li> Preparare 1: 1.000 diluizioni di anticorpi secondari Alexa-Fluor in 0,5% Triton X-100,10% di siero asino in PBS e conservare al buio. </li><li> Rimuovere delicatamente la PBS risciacquo finale e applicare la diluizione anticorpo secondario per un&#39;ora al buio. </li><li> Rimuovere la soluzione anticorpo secondario, sciacquare e lavare la sezione tre volte. Se necessario, applicare una colorazione nucleare, come DAPI (~ 300nm), durante la prima applicazione PBS. </li><li> Rimuovere completamente la soluzione di PBS da tutto il tessuto e posto ~ 5-7 ml di mezzo di montaggio sulla sezione di tessuto. Collocare un vetrino di vetro sopra la sezione di tessuto e ritirate ~ 15-30 minuti a temperatura ambiente al buio. </li><li> Utilizzare un microscopio a fluorescenza dotato i filtri appropriati per visualizzare EGFP e qualsiasi altre lunghezze d&#39;onda appropriate per gli anticorpi secondari usati nelle fasi 5.8 / 5.9. A fette cerebellare, utilizzare un obiettivo 20X per visualizzare cerchi nucleari EGFP-KASH2. </li></ol>

<ol>
	<li>Hutchison, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamins:+building+blocks+or+regulators+of+gene+expression?+Nature+reviews.">Lamins: building blocks or regulators of gene expression? Nature reviews.</a> Molecular cell biology. 3, 848-858 (2002).</li><li>Stuurman, N., Heins, S., Aebi, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+lamins:+their+structure,+assembly,+and+interactions.">Nuclear lamins: their structure, assembly, and interactions.</a> Journal of structural biology. 122, 42-66 (1998).</li><li>Crisp, 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=Coupling+of+the+nucleus+and+cytoplasm:+role+of+the+LINC+complex.">Coupling of the nucleus and cytoplasm: role of the LINC complex.</a> The Journal of cell biology. 172, 41-53 (2006).</li><li>Haque, F., 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=SUN1+interacts+with+nuclear+lamin+A+and+cytoplasmic+nesprins+to+provide+a+physical+connection+between+the+nuclear+lamina+and+the+cytoskeleton.">SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton.</a> Molecular and cellular biology. 26, 3738-3751 (2006).</li><li>Hodzic, D. M., Yeater, D. B., Bengtsson, L., Otto, H., Stahl, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sun2+is+a+novel+mammalian+inner+nuclear+membrane+protein.">Sun2 is a novel mammalian inner nuclear membrane protein.</a> The Journal of biological chemistry. 279, 25805-25812 (2004).</li><li>Starr, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KASH+and+SUN+proteins.">KASH and SUN proteins.</a> Current biology : CB. 21, R414-R415 (2011).</li><li>Mellad, J. A., Warren, D. T., Shanahan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nesprins+LINC+the+nucleus+and+cytoskeleton.">Nesprins LINC the nucleus and cytoskeleton.</a> Current opinion in cell biology. 23, 47-54 (2011).</li><li>Fridolfsson, H. N., Starr, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-1+and+dynein+at+the+nuclear+envelope+mediate+the+bidirectional+migrations+of+nuclei.">Kinesin-1 and dynein at the nuclear envelope mediate the bidirectional migrations of nuclei.</a> The Journal of cell biology. 191, 115-128 (2010).</li><li>Meyerzon, M., Fridolfsson, H. N., Ly, N., McNally, F. J., Starr, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNC-83+is+a+nuclear-specific+cargo+adaptor+for+kinesin-1-mediated+nuclear+migration.">UNC-83 is a nuclear-specific cargo adaptor for kinesin-1-mediated nuclear migration.</a> Development. 136, 2725-2733 (2009).</li><li>Padmakumar, V. 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=Enaptin,+a+giant+actin-binding+protein,+is+an+element+of+the+nuclear+membrane+and+the+actin+cytoskeleton.">Enaptin, a giant actin-binding protein, is an element of the nuclear membrane and the actin cytoskeleton.</a> Experimental cell research. 295, 330-339 (2004).</li><li>Roux, K. 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=Nesprin+4+is+an+outer+nuclear+membrane+protein+that+can+induce+kinesin-mediated+cell+polarization.">Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 2194-2199 (2009).</li><li>Wilhelmsen, 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=a+novel+outer+nuclear+membrane+protein,+associates+with+the+cytoskeletal+linker+protein+plectin.">a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin.</a> The Journal of cell biology. 171, 799-810 (2005).</li><li>Wilson, M. H., Holzbaur, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nesprins+anchor+kinesin-1+motors+to+the+nucleus+to+drive+nuclear+distribution+in+muscle+cells.">Nesprins anchor kinesin-1 motors to the nucleus to drive nuclear distribution in muscle cells.</a> Development. 142, 218-228 (2015).</li><li>Yu, 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=KASH+protein+Syne-2/Nesprin-2+and+SUN+proteins+SUN1/2+mediate+nuclear+migration+during+mammalian+retinal+development.">KASH protein Syne-2/Nesprin-2 and SUN proteins SUN1/2 mediate nuclear migration during mammalian retinal development.</a> Human molecular genetics. 20, 1061-1073 (2011).</li><li>Zhang, X., 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=Syne-1+and+Syne-2+play+crucial+roles+in+myonuclear+anchorage+and+motor+neuron+innervation.">Syne-1 and Syne-2 play crucial roles in myonuclear anchorage and motor neuron innervation.</a> Development. 134, 901-908 (2007).</li><li>Zhang, 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=Nesprin+1+is+critical+for+nuclear+positioning+and+anchorage.">Nesprin 1 is critical for nuclear positioning and anchorage.</a> Human molecular genetics. 19, 329-341 (2010).</li><li>Stewart-Hutchinson, P. J., Hale, C. M., Wirtz, D., Hodzic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+requirements+for+the+assembly+of+LINC+complexes+and+their+function+in+cellular+mechanical+stiffness.">Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness.</a> Experimental cell research. 314, 1892-1905 (2008).</li><li>Zhang, X., 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=SUN1/2+and+Syne/Nesprin-1/2+complexes+connect+centrosome+to+the+nucleus+during+neurogenesis+and+neuronal+migration+in+mice.">SUN1/2 and Syne/Nesprin-1/2 complexes connect centrosome to the nucleus during neurogenesis and neuronal migration in mice.</a> Neuron. 64, 173-187 (2009).</li><li>Razafsky, D., Hodzic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+tissue-specific+disruption+of+LINC+complexes+in+vivo.">Temporal and tissue-specific disruption of LINC complexes in vivo.</a> Genesis. 52, 359-365 (2014).</li><li>Razafsky, D., Hodzic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+Nesprin1+giant+devoid+of+KASH+domain+underlies+the+molecular+etiology+of+autosomal+recessive+cerebellar+ataxia+type+I.">A variant of Nesprin1 giant devoid of KASH domain underlies the molecular etiology of autosomal recessive cerebellar ataxia type I.</a> Neurobiology of disease. 78, 57-67 (2015).</li><li>Razafsky, D., Blecher, N., Markov, A., Stewart-Hutchinson, P. J., Hodzic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LINC+complexes+mediate+the+positioning+of+cone+photoreceptor+nuclei+in+mouse+retina.">LINC complexes mediate the positioning of cone photoreceptor nuclei in mouse retina.</a> PloS one. 7, e47180 (2012).</li><li>Razafsky, D. S., Ward, C. L., Kolb, T., Hodzic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+linkers+of+the+nucleoskeleton+to+the+cytoskeleton+during+mouse+postnatal+retinogenesis.">Developmental regulation of linkers of the nucleoskeleton to the cytoskeleton during mouse postnatal retinogenesis.</a> Nucleus. 4, 399-409 (2013).</li></ol>

Gli autori non hanno nulla da rivelare.

La fase più critica per studiare con successo il ruolo di complessi LINC in vivo utilizzando la (/ EGFP-KASH2 CAG-LacZ) modello Tg è quello di individuare una linea di topi Cre adatto (s). Infatti, se Cre è attiva in altri tipi di cellule coinvolte in percorsi simili, può complicare l&#39;interpretazione dei risultati. Quindi, è importante esaminare cellule vicine, come mostrato qui negli strati cellulari e molecolari granuli del cervelletto (Figura 2B). Analogamente, per massimizzare even...

L&#39;involucro nucleare (NE) separa nucleoplasma dal citoplasma. Esso è composto da una membrana nucleare interna ed esterna (INM e ONM, rispettivamente) che collegano a pori nucleari. Il lume delineato da due membrane si chiama lo spazio perinucleare (PNS). Il ONM è un&#39;estensione del reticolo endoplasmatico rugoso (ER) e l&#39;INM aderisce alla lamina nucleare, un reticolo di nucleare tipo V filamenti intermedi rappresentato da A e B lamine di tipo 1,2. Linker del nucleoscheletro e citoscheletro (LINC) sono complessi macromolecolari assemblee che attraversano l&#39;intero involucro nucleare collegare fisicamente l&#39;interno del nucleo di filamenti citoscheletrici e motori molecolari (Figura 1A). Sono costituiti da interazioni tra motivi evolutivamente conservati che caratterizzano due famiglie di proteine ​​transmembrana integranti del NE: Sun (SAD1 / Unc84) proteine ​​e Nesprins (nucleare Envelope SPectRINS). Nei mammiferi, Sun1 e Sun2 are transmembrana proteine ​​della INM la cui regione nucleoplasmic N-terminale interagisce direttamente con A e B lamine di tipo 3-5. Sull&#39;altro lato della INM, all&#39;interno del PNS, proteine ​​Sun Harbor un tratto evolutivo conservata di ~ 150 amminoacidi C-terminale chiamato dominio SUN. Domini SUN interagiscono direttamente con il evolutivo conservata KASH (Klarsicht / Anc-1, Syne omologia) dominio, la firma molecolare di Nesprins. Domini KASH consistono di un tratto di ~ 30 C-terminale amminoacidi che sporge nel PNS seguito da un dominio transmembrana 6. Almeno quattro geni distinti Nesprin (Nesprin1-4) codificano KASH contenenti proteine ​​che localizzano al NE 7. Le regioni citoplasmatici di Nesprins, il cui variare taglie da ~ 50kDa (Nesprin4) a un sorprendente 1.000 kDa (Nesprin1 gigante), contengono spectrina multiplo ripete così come i motivi specifici che consente loro interazione con componenti del citoscheletro, come l&#39;actina, plectina e motori molecolari 8- 13. &lt;p class = &quot;jove_content&quot;&gt; Studi di vertebrati e invertebrati hanno mostrato che / motori molecolari Lamin Sole / Nesprin / costituiscono un &quot;asse&quot; evolutivamente conservata controllare la migrazione nucleare e di ancoraggio. Diversi knock-out (KO) modelli murini di componenti complessi LINC sono stati descritti e sono stati strumentali nel fornire un quadro per capire il ruolo delle proteine ​​Sole e Nesprin a NE durante lo sviluppo dei mammiferi 9,14,15. Tuttavia, questi modelli presentano diversi svantaggi significativi, più in particolare: 1) difficoltà nell&#39;interpretare fenotipi dovuti alla cella effetti non autonomi, 2) difficoltà nel distinguere i contributi fenotipiche di Kash contenenti vs. KASH-meno Nesprin isoforme 16, 3) il la ridondanza funzionale delle proteine ​​Sole e Nesprin al NE in numerosi tipi di cellule richiede sistemi di allevamento complessi per inattivare tutte le interazioni SUN-Kash in topi 17 e 4) la letalità perinatale di topi deficienti per la KASH-dominio di entrambiNesprins1 e 2 preclude l&#39;analisi dei fenotipi adulti 18. Questo protocollo descrive un modello di topo romanzo progettata per distruggere tutte le interazioni SUN-KASH in vivo, in una cella modo autonomo e evolutivamente regolata, bypassando così molti degli inconvenienti sopra delineati. Questo / modello murino basato LOX-Cre si basa su due concetti importanti: 1) il dominio KASH di qualsiasi proteina Nesprin noto è sufficiente per indirizzare EGFP a NE in sistemi di colture cellulari e 2) i domini SUN interagire promiscuamente con i domini Kash, quindi sovraespressione di qualsiasi dominio KASH saturerà tutti i domini SUN endogeni e inattivare complessi LINC in modo negativamente dominante 17 (Figura 1B). Questo protocollo descrive la raccolta dei tessuti e fasi di lavorazione utilizzate per confermare l&#39;interruzione di tutte le interazioni SUN-Kash in cellule di Purkinje cerebellari.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion Slides</td>
			<td>StatLab</td>
			<td>M1000W</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Sigma Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmuEdge Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Calbindin Antibody</td>
			<td>Sigma Aldrich</td>
			<td>C9848</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-EGFP Antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nesprin2 Antibody</td>
			<td></td>
			<td></td>
			<td>Previously described in Ref. 21</td>
		</tr>
		<tr>
			<td>Fluorescent Mounting Media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>Sigma Aldrich</td>
			<td>O3551</td>
			<td></td>
		</tr>
	</tbody>
</table>

validation of a mouse model to disrupt linc complexes in a cell-specific manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Questo protocollo illustra l&#39;utilità del (/ EGFP-KASH2 CAG-LacZ) modello di topo Tg di limitare l&#39;espressione EGFP-KASH2 alle cellule di Purkinje cerebellari utilizzando Tg (PCP2-Cre) topi. Nel Tg (PCP2Cre CAG-EGFP / KASH2) prole, la griglia di lettura aperta LacZ / V5 è asportato in P6 dalla ricombinasi Cre determinando così l&#39;espressione di EGFP-KASH2 specificamente mirato al Purkinje cellule (Figura 2A). Come previsto, EGFP-KASH2 è rivolto a membrana nucleare come indicato ...

Watch this Scientific Journal Video about Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner at JoVE.com

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

La convalida di un modello di topo per Disrupt LINC Complessi in modo specifico-Cell

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

validation-mouse-model-to-disrupt-linc-complexes-cell-specific

Research

JoVE Journal

Biology

7.3K Views.  Washington University in St. Louis School of Medicine. Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Video: Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

L&#39;applicazione di una degenerazione C. elegans Dopamina Saggio Neuron per la convalida dei potenziali morbo di Parkinson geni

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Ricerca

Biologia

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-inner Cell Mass iniezione delle cellule staminali embrionali porta a tassi più elevati di chimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Un'analisi di citometria-basata di flusso per identificare i composti che interrompono il grippaggio del Ligand di Chemokine CXC fluorescente etichettati 12 al recettore di chemochine CXC 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Un modello murino di ostruzione intestinale parziale

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Analisi della spettrometria di massa per identificare l'ubiquilazione del CENP-A con tag EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Un modello di topo di instabilità della colonna lombare

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Adipociti di topo differenziati in coltura primaria: un modello di insulino-resistenza

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...