The authors thank the staff of the Morphology and Imaging core, of the Molecular Genetics core (Department of Ophthalmology and Visual Sciences) and of the Mouse Genetics Core at Washington University in St. Louis School of Medicine. The authors are supported by the small grant program from The McDonnell Center for Cellular and Molecular Neurobiology, the Hope Center for Neurological Disorders, the National Eye Institute (#R01EY022632 to D.H.), a National Eye Institute Center Core Grant (#P30EY002687) and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology and Visual Sciences.

Ethics Statement: Procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee (IACUC) at Washington University in St. Louis.
1. Mouse Breeding and Genotyping
<ol>
	<li>Breed Tg(CAG-LacZ/EGFP-KASH2) mice with Tg(PCP2-Cre) mice to produce Tg(PCP2CreCAG-EGFP/KASH2) 19,20. Note: While the remainder of this protocol focuses on the use of the Tg(PCP2-Cre) mouse line to restrict EGFP-KASH2 expression to Purkinje cells within the cerebellum, a similar procedure can be followed with any other Tg(Cre) mouse (Figure 2A).</li>
	<li>Perform genotyping to identify pups harboring both EGFP and Cre transgenes as previously described 19.</li>
</ol>
2. Preparation of Materials for Dissection and Tissue Collection
<ol>
	<li>Prepare 100 ml of 30% sucrose in PBS (30 g sucrose/100 ml of 1x PBS) and store at -20&#176;C. The day of tissue harvesting, thaw and load ~30-35 ml of sucrose solution into a 35 ml syringe for transcardial perfusions.</li>
	<li>Prepare 40 ml of 4% PFA in PBS (10 ml 16% paraformaldehyde and 30 ml PBS). Load ~30-35 ml of 4% PFA into a 35 ml syringe for transcardial perfusions.</li>
	<li>Fill a 10 cm tissue culture plate with 10 ml of PBS for tissue dissection.</li>
	<li>Prepare a ketamine/xylazine cocktail consisting of 16 mg/ml ketamine and 3 mg/ml xylazine.</li>
	<li>Prepare a solution of 70% ethanol for the dissection.</li>
</ol>
3. Dissection and Tissue Collection
<ol>
	<li>Using a 1 ml syringe, inject 500 &#181;l of the ketamine/xylazine cocktail per 25 grams of body weight and wait for ~15 min. Alternatively, use another institutionally approved sedation method that is appropriate for the biological system of interest.</li>
	<li>Upon sedation, gently pinch the rear paw to confirm the lack of pain response, pin all paws on a Styrofoam tray with the dorsal side of the mouse facing down.</li>
	<li>Spray the abdomen with 70% ethanol and make an incision from the lower abdomen to the top of the rib cage to gain access to the heart.</li>
	<li>Make a small incision in the right atrium of the heart and insert the syringe containing the 30% sucrose solution into the left ventricle of the heart.</li>
	<li>Perfuse the solution at a rate of ~2-5 ml per min. When empty, remove the syringe and repeat with the 4% PFA solution.</li>
	<li>Unpin the mouse from the Styrofoam tray and decapitate to access the brain.</li>
	<li>Remove the skin around the head of the animal using forceps and scissors. Poke a hole in the posterior portion of the skull with a needle and carefully remove the skull with scissors and dissecting forceps.</li>
	<li>Once the brain is clearly accessible, use forceps to remove the cerebellum and place it in PBS. Bisect the cerebellum in the sagittal plane with a scalpel and transfer to the 30% sucrose solution overnight at 4&#176;C. Note: To prepare paraffin sections, fix the cerebellum overnight at 4&#176;C in 4% PFA prior to tissue embedding and sectioning.</li>
</ol>
4. Tissue Processing and Sectioning
<ol>
	<li>Prepare a slurry of dry ice and 2-methyl butane. Allow the temperature of the bath to equilibrate for 5 min.</li>
	<li>Fill a cryomold with OCT compound and place one half of the bisected cerebellum into the mold.</li>
	<li>Using a dissecting microscope and a pair of forceps position the cerebellum so that the bisected surface (near the midline of the cerebellum) is facing up.</li>
	<li>Transfer the cryomold to the dry ice slurry for 5 min until the OCT compound has formed a solid white matrix around the tissue specimen. Note: The frozen specimen can now be stored at -20&#176;C if required.</li>
	<li>Label pre-treated poly-lysine slides for collecting tissue sections and prepare the cryostat according to the manufacturer&#39;s instructions in order to collect 15 &#181;m sections of the cerebellum. To collect the sections, carefully contact the OCT section with the poly-lysine coated side of the slide. To preserve the orientation of the section, do not rotate the slide during this step.</li>
	<li>Store tissue sections at -20&#176;C until proceeding with staining and imaging.</li>
</ol>
5. Staining and Imaging of Cerebellum Sections
<ol>
	<li>Prepare the following solutions prior to tissue staining: 
	~50 ml&#160; of PBS 
	~5-10 ml&#160; of 4% PFA in PBS 
	~10-20 ml&#160; of 0.5% Triton X-100, 10% donkey serum in PBS</li>
	<li>Thaw cerebellar sections. Importantly, minimize their exposure to any source of continuous and intense light to prevent fading of EGFP.</li>
	<li>Using a hydrophobic barrier pen, draw a rectangle around the tissue sections.</li>
	<li>Post-fix the sections with 4% PFA for 5 min. Use enough liquid to cover the entire area delineated by the hydrophobic barrier (usually ~500 &#181;l). Using a vacuum or pipette, remove the fixative being particularly careful not to disturb the tissue sections.</li>
	<li>With a pipette, gently apply PBS on the slide without disturbing the tissue sections. After 5 min use the vacuum or pipette to carefully remove the liquid. Repeat the PBS rinse a total of three times.</li>
	<li>Using a pipette, carefully apply the appropriate dilution of primary antibodies (3 &#181;l Calbindin and/or Nesprin2 antibody per 900 &#181;l of solution) in 0.5% Triton X-100, 10% donkey serum in PBS. Incubate for one hour at room temperature in the dark. 
	Note: EGFP-KASH2 will be visible by direct fluorescence microscopy on frozen sections, and it is not necessary to utilize an anti-EGFP antibody. However, if paraffin sections have been prepared, imaging of EGFP-KASH2 requires immunodetection with an anti-EGFP antibody.</li>
	<li>Remove the antibody solution and rinse with PBS three times.</li>
	<li>Prepare 1:1,000 dilutions of Alexa-Fluor secondary antibodies in 0.5% Triton X-100, 10% donkey serum in PBS and store in the dark.</li>
	<li>Gently remove the final PBS rinse and apply the secondary antibody dilution for one hour in the dark.</li>
	<li>Remove the secondary antibody solution, rinse and wash the section three times. If needed, apply a nuclear stain, such as DAPI (~300nM), during the first PBS application.</li>
	<li>Completely remove the PBS solution from around the tissue and place ~5-7 &#181;l of mounting media on the tissue section. Place a glass coverslip on top of the tissue section and set aside for ~15-30 min at room temperature in the dark.</li>
	<li>Use a fluorescence microscope equipped with the appropriate filters to visualize EGFP and any other wavelengths appropriate for the secondary antibodies used in steps 5.8/5.9. In cerebellar slices, use a 20X objective to view EGFP-KASH2 nuclear rims.</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>

The authors have nothing to disclose.

The most critical step to successfully study the role of LINC complexes in vivo using the Tg(CAG-LacZ/EGFP-KASH2) model is to identify a suitable Cre mouse line(s). Indeed, if Cre is active in other cell-types involved in similar pathways, it can complicate the interpretation of the results. Hence, it is important to examine nearby cells, as shown here in the molecular and granule cell layers of the cerebellum (Figure 2B). Likewise, to maximize any physiologically relevant phenotypes it is impor...

The nuclear envelope (NE) separates the nucleoplasm from the cytoplasm. It is composed of an inner and outer nuclear membrane (INM and ONM, respectively) that connect at nuclear pores. The lumen delineated by both membranes is called the perinuclear space (PNS). The ONM is an extension of the rough endoplasmic reticulum (ER), and the INM adheres to the nuclear lamina, a meshwork of nuclear type-V intermediate filaments represented by A- and B-type lamins 1,2. LInkers of the Nucleoskeleton and Cytoskeleton (LINC) complexes are macromolecular assemblies that span the whole nuclear envelope to physically connect the interior of the nucleus to cytoskeletal filaments and molecular motors (Figure 1A). They consist of interactions between evolutionarily conserved motifs that characterize two families of integral transmembrane proteins of the NE: Sun (Sad1/Unc84) proteins and Nesprins (Nuclear Envelope SPectRINS). In mammals, Sun1 and Sun2 are transmembrane proteins of the INM whose N-terminal nucleoplasmic region interacts directly with A- and B-type lamins 3-5. On the other side of the INM, within the PNS, Sun proteins harbor an evolutionary-conserved stretch of ~150 C-terminal amino acids called the SUN domain. SUN domains interact directly with the evolutionary-conserved KASH (Klarsicht/Anc-1, Syne Homology) domain, the molecular signature of Nesprins. KASH domains consist of a stretch of ~30 C-terminal amino acids that protrudes into the PNS followed by a transmembrane domain 6. At least four distinct Nesprin genes (Nesprin1-4) encode KASH-containing proteins that localize at the NE 7. The cytoplasmic regions of Nesprins, whose sizes vary from ~50kDa (Nesprin4) to an astonishing 1,000 kDa (Nesprin1 giant), contain multiple spectrin repeats as well as specific motifs enabling their interaction with cytoskeletal components such as actin, plectin and molecular motors8-13.
Studies in vertebrates and invertebrates have shown that Lamin/Sun/Nesprin/molecular motors constitute an evolutionarily conserved &#8220;axis&#8221; controlling nuclear migration and anchorage. Several knock-out (KO) mouse models of LINC complex components have been described and were instrumental in providing a framework to understand the roles of Sun and Nesprin proteins at the NE during mammalian development 9,14,15. However, these models present several significant drawbacks, most notably: 1) difficulty in interpreting phenotypes due to cell non-autonomous effects, 2) difficulty in distinguishing the phenotypical contributions of KASH-containing vs. KASH-less Nesprin isoforms16, 3) the functional redundancy of Sun and Nesprin proteins at the NE in numerous cell types requires complex breeding schemes to inactivate all SUN-KASH interactions in mice 17 and 4) the perinatal lethality of mice deficient for the KASH-domain of both Nesprins1 and 2 precludes the analysis of adult phenotypes 18.
This protocol describes a novel mouse model designed to disrupt all SUN-KASH interactions in&#160;vivo, in a cell autonomous and developmentally regulated manner, thus bypassing many of the drawbacks outlined above. This Cre/lox-based mouse model relies on two important concepts: 1) the KASH domain of any known Nesprin protein is sufficient to target EGFP to the NE in cell culture systems and 2) SUN domains interact promiscuously with KASH domains, thus overexpression of any KASH domain will saturate all endogenous SUN domains and inactivate LINC complexes in a dominant-negative manner 17 (Figure 1B). This protocol describes tissue harvesting and processing steps used to confirm the disruption of all SUN-KASH interactions in cerebellar Purkinje cells.

<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.

This protocol illustrates the usefulness of the Tg(CAG-LacZ/EGFP-KASH2) mouse model to restrict EGFP-KASH2 expression to cerebellar Purkinje cells using Tg(PCP2-Cre) mice. In Tg(PCP2CreCAG-EGFP/KASH2) offspring, the LacZ/V5 open reading frame is excised at P6 by the Cre recombinase thereby leading to the expression of EGFP-KASH2 specifically targeted to Purkinje cells (Figure 2A). As expected, EGFP-KASH2 is targeted to the nuclear envelope as indicated by the EGFP-positive rim-like pattern obs...

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.

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.

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

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.

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.

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.

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).

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.

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.

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.

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.

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