Name: validation of a mouse model to disrupt linc complexes in a cell-specific manner
Uploaded: 2015-12-10T13:00:00
Description: Watch this Scientific Journal Video about Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner at JoVE.com

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 espres...

<ol>
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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>

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 ...

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