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/pubmed?term=((Lamins%3A+building+blocks+or+regulators+of+gene+expression%3F+Nature+reviews%5BTitle%5D)+AND+%22Molecular+cell+biology%22%5BJournal%5D)">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/pubmed?term=((Nuclear+lamins%3A+their+structure%2C+assembly%2C+and+interactions%5BTitle%5D)+AND+%22Journal+of+structural+biology%22%5BJournal%5D)">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/pubmed?term=((Coupling+of+the+nucleus+and+cytoplasm%3A+role+of+the+LINC+complex%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">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/pubmed?term=((SUN1+interacts+with+nuclear+lamin+A+and+cytoplasmic+nesprins+to+provide+a+physical+connection+between+the+nuclear+lamina+and+the+cytoskeleton%5BTitle%5D)+AND+%22Molecular+and+cellular+biology%22%5BJournal%5D)">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/pubmed?term=((Sun2+is+a+novel+mammalian+inner+nuclear+membrane+protein%5BTitle%5D)+AND+%22The+Journal+of+biological+chemistry%22%5BJournal%5D)">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/pubmed?term=((KASH+and+SUN+proteins%5BTitle%5D)+AND+%22Current+biology+%3A+CB%22%5BJournal%5D)">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/pubmed?term=((Nesprins+LINC+the+nucleus+and+cytoskeleton%5BTitle%5D)+AND+%22Current+opinion+in+cell+biology%22%5BJournal%5D)">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/pubmed?term=((Kinesin-1+and+dynein+at+the+nuclear+envelope+mediate+the+bidirectional+migrations+of+nuclei%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">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/pubmed?term=((UNC-83+is+a+nuclear-specific+cargo+adaptor+for+kinesin-1-mediated+nuclear+migration%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Enaptin%2C+a+giant+actin-binding+protein%2C+is+an+element+of+the+nuclear+membrane+and+the+actin+cytoskeleton%5BTitle%5D)+AND+%22Experimental+cell+research%22%5BJournal%5D)">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/pubmed?term=((Nesprin+4+is+an+outer+nuclear+membrane+protein+that+can+induce+kinesin-mediated+cell+polarization%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((a+novel+outer+nuclear+membrane+protein%2C+associates+with+the+cytoskeletal+linker+protein+plectin%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">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/pubmed?term=((Nesprins+anchor+kinesin-1+motors+to+the+nucleus+to+drive+nuclear+distribution+in+muscle+cells%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((KASH+protein+Syne-2%2FNesprin-2+and+SUN+proteins+SUN1%2F2+mediate+nuclear+migration+during+mammalian+retinal+development%5BTitle%5D)+AND+%22Human+molecular+genetics%22%5BJournal%5D)">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/pubmed?term=((Syne-1+and+Syne-2+play+crucial+roles+in+myonuclear+anchorage+and+motor+neuron+innervation%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Nesprin+1+is+critical+for+nuclear+positioning+and+anchorage%5BTitle%5D)+AND+%22Human+molecular+genetics%22%5BJournal%5D)">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/pubmed?term=((Structural+requirements+for+the+assembly+of+LINC+complexes+and+their+function+in+cellular+mechanical+stiffness%5BTitle%5D)+AND+%22Experimental+cell+research%22%5BJournal%5D)">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/pubmed?term=((SUN1%2F2+and+Syne%2FNesprin-1%2F2+complexes+connect+centrosome+to+the+nucleus+during+neurogenesis+and+neuronal+migration+in+mice%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((Temporal+and+tissue-specific+disruption+of+LINC+complexes+in+vivo%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">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/pubmed?term=((A+variant+of+Nesprin1+giant+devoid+of+KASH+domain+underlies+the+molecular+etiology+of+autosomal+recessive+cerebellar+ataxia+type+I%5BTitle%5D)+AND+%22Neurobiology+of+disease%22%5BJournal%5D)">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/pubmed?term=((LINC+complexes+mediate+the+positioning+of+cone+photoreceptor+nuclei+in+mouse+retina%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">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/pubmed?term=((Developmental+regulation+of+linkers+of+the+nucleoskeleton+to+the+cytoskeleton+during+mouse+postnatal+retinogenesis%5BTitle%5D)+AND+%22Nucleus%22%5BJournal%5D)">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 important to identify a Cre strain with widespread Cre expression across the cell type of interest. It is also vital to undertake an in-depth characterization of the expression pattern of LINC complex components during the development of the system of interest. Indeed, one drawback of the Tg(CAG-LacZ/EGFP-KASH2) mouse model is that it does not provide any information as to which Sun proteins or Nesprins variants are involved in the biological system under consideration. However, this issue can be overcome by undertaking a rigorous characterization at both the transcript and protein levels. In fact, such studies have been undertaken on several occasions which led to important findings about the role of specific LINC complex components during CNS development and homeostasis21,22.
Finally, while this protocol is focused on the use of fixed CNS tissues, Tg(CAG-LacZ/EGFP-KASH2) mice can be used to isolate and culture primary cells to study the effect of disrupting LINC complexes in vitro. Using this scheme, it is possible to isolate and purify primary cultures of EGFP-KASH2-positive cells for applications such as time-lapse video microscopy. This bypasses low transfection efficiencies or lentiviral production required for primary culture systems, such as neurons.
In summary, the Tg(CAG-LacZ/EGFP-KASH2) mice open up new and exciting possibilities to study the function of LINC complexes in vivo during embryonic and postnatal mammalian developmental processes in a wide variety of tissues and cell types to address basic cell biology questions.

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><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 observed around the nucleus (Figure 2B). To maximize any physiological phenotype, it is important to ensure that EGFP-KASH2 is expressed in a majority of targeted cells by calculating the percentage of EGFP-KASH2 positive cells that are co-stained with a cell-specific antibody marker. In this example, EGFP-KASH2 expression was detected in more than 70% of Calbindin-positive Purkinje cells (Figure 2B). In these conditions, we did not observe any significant histological or behavioral phenotypes in 10 month-old Tg(PCP2CreCAG-EGFP/KASH2) mice20. It is important to ensure that any morphological or behavioral phenotype is not due to ectopic expression of EGFP-KASH2 in an unintended cell type/tissue. To that respect, EGFP-KASH2 was not observed in the granule cell layer or the molecular layer of Tg(PCP2CreCAG-EGFP-KASH2) cerebella (Figure 2B).
Finally, it is vital to confirm the extent of LINC complex disruption in cells expressing EGFP-KASH2. This can be achieved using high-quality anti-Nesprin antibodies to emphasize their displacement from the NE of cells expressing EGFP-KASH2. As shown in Figure 3, endogenous Nesprin2 is displaced from the nuclear envelope of Purkinje cells that express EGFP-KASH2 (Figure 3 top panel, yellow arrows) while Purkinje cells that do not express EGFP-KASH2 retain endogenous Nesprin2 at the nuclear envelope (Figure 3 top panel, white arrow). As expected, control littermates, which do not express Cre-recombinase, display Nesprin2 at the nuclear envelope of the whole Purkinje cell population (Figure 3 bottom white arrows). Several reports have now confirmed the expression of EGFP-KASH2, accompanied by the displacement of endogenous Nesprins, in additional tissues and cell types such as skeletal muscle fibers and cone photoreceptors19,21.
<img alt="Figure 1" src="/files/ftp_upload/53318/53318fig1.jpg" /> 
Figure 1.&#160;LINC complexes connect the nucleus to the cytoskeleton and are disrupted by the overexpression of EGFP-KASH2.&#160;(A) Depiction of LINC complexes that connect the interior of the nucleus to the cytoplasmic cytoskeleton. Note that SUN domains interact promiscuously with KASH domains from all Nesprins (Nesprins 1-4). (B) Overexpression of EGFP-KASH2 displaces endogenous Nesprins from the nuclear envelope to the ER, in a dominant negative fashion, thus disconnecting the nucleus from the surrounding cytoplasmic cytoskeleton.
<img alt="Figure 2" src="/files/ftp_upload/53318/53318fig2.jpg" /> 
Figure 2.&#160;Overexpression of EGFP-KASH2 specifically targeted to cerebellar Purkinje cells.&#160;(A) Breeding of Tg(CAG-LacZ/EGFP-KASH2) to Tg(PCP2-Cre) mice results in Tg(PCP2CreCAG-EGFP-KASH2) offspring in which Cre recombinase is expressed specifically in cerebellar Purkinje cells. Upon expression (P6), Cre recombinase translocates into the nucleus where it excises the LacZ ORF and induces EGFP-KASH2 expression. (B) Tg(PCP2CreCAG-EGFP-KASH2) mice specifically express EGFP-KASH2 in cerebellar Purkinje cells identified with Calbindin (colored in red). Note that both cerebellar granule neurons and interneurons of the molecular layer are negative for EGFP-KASH2 expression. Abbreviations: MoL: Molecular layer, PCL: Purkinje cell layer and GCL: Granule cell layer.
<img alt="Figure 3" src="/files/ftp_upload/53318/53318fig3.jpg" /> 
Figure 3.&#160;Disruption of endogenous LINC complexes in cerebellar Purkinje cells. Single confocal plane showing expression of EGFP-KASH2 that displaces endogenous Nesprin2 (colored in red) from the nuclear envelope of Purkinje cells (yellow arrows top). Note that Purkinje cells lacking EGFP-KASH2 expression maintain endogenous Nesprin2 at their nuclear envelope (white arrow top). In Tg(CAG-LacZ/EGFP-KASH2) control littermates, no EGFP signal is observed and Nesprin2 is detected at the nuclear envelope of all Purki
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 observed around the nucleus (Figure 2B). To maximize any physiological phenotype, it is important to ensure that EGFP-KASH2 is expressed in a majority of targeted cells by calculating the percentage of EGFP-KASH2 positive cells that are co-stained with a cell-specific antibody marker. In this example, EGFP-KASH2 expression was detected in more than 70% of Calbindin-positive Purkinje cells (Figure 2B). In these conditions, we did not observe any significant histological or behavioral phenotypes in 10 month-old Tg(PCP2CreCAG-EGFP/KASH2) mice20. It is important to ensure that any morphological or behavioral phenotype is not due to ectopic expression of EGFP-KASH2 in an unintended cell type/tissue. To that respect, EGFP-KASH2 was not observed in the granule cell layer or the molecular layer of Tg(PCP2CreCAG-EGFP-KASH2) cerebella (Figure 2B).
Finally, it is vital to confirm the extent of LINC complex disruption in cells expressing EGFP-KASH2. This can be achieved using high-quality anti-Nesprin antibodies to emphasize their displacement from the NE of cells expressing EGFP-KASH2. As shown in Figure 3, endogenous Nesprin2 is displaced from the nuclear envelope of Purkinje cells that express EGFP-KASH2 (Figure 3 top panel, yellow arrows) while Purkinje cells that do not express EGFP-KASH2 retain endogenous Nesprin2 at the nuclear envelope (Figure 3 top panel, white arrow). As expected, control littermates, which do not express Cre-recombinase, display Nesprin2 at the nuclear envelope of the whole Purkinje cell population (Figure 3 bottom white arrows). Several reports have now confirmed the expression of EGFP-KASH2, accompanied by the displacement of endogenous Nesprins, in additional tissues and cell types such as skeletal muscle fibers and cone photoreceptors19,21.
<img alt="Figure 1" src="/files/ftp_upload/53318/53318fig1.jpg" /> 
Figure 1.&#160;LINC complexes connect the nucleus to the cytoskeleton and are disrupted by the overexpression of EGFP-KASH2.&#160;(A) Depiction of LINC complexes that connect the interior of the nucleus to the cytoplasmic cytoskeleton. Note that SUN domains interact promiscuously with KASH domains from all Nesprins (Nesprins 1-4). (B) Overexpression of EGFP-KASH2 displaces endogenous Nesprins from the nuclear envelope to the ER, in a dominant negative fashion, thus disconnecting the nucleus from the surrounding cytoplasmic cytoskeleton. <a href="https://www.jove.com/files/ftp_upload/53318/53318fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53318/53318fig2.jpg" /> 
Figure 2.&#160;Overexpression of EGFP-KASH2 specifically targeted to cerebellar Purkinje cells.&#160;(A) Breeding of Tg(CAG-LacZ/EGFP-KASH2) to Tg(PCP2-Cre) mice results in Tg(PCP2CreCAG-EGFP-KASH2) offspring in which Cre recombinase is expressed specifically in cerebellar Purkinje cells. Upon expression (P6), Cre recombinase translocates into the nucleus where it excises the LacZ ORF and induces EGFP-KASH2 expression. (B) Tg(PCP2CreCAG-EGFP-KASH2) mice specifically express EGFP-KASH2 in cerebellar Purkinje cells identified with Calbindin (colored in red). Note that both cerebellar granule neurons and interneurons of the molecular layer are negative for EGFP-KASH2 expression. Abbreviations: MoL: Molecular layer, PCL: Purkinje cell layer and GCL: Granule cell layer. <a href="https://www.jove.com/files/ftp_upload/53318/53318fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53318/53318fig3.jpg" /> 
Figure 3.&#160;Disruption of endogenous LINC complexes in cerebellar Purkinje cells. Single confocal plane showing expression of EGFP-KASH2 that displaces endogenous Nesprin2 (colored in red) from the nuclear envelope of Purkinje cells (yellow arrows top). Note that Purkinje cells lacking EGFP-KASH2 expression maintain endogenous Nesprin2 at their nuclear envelope (white arrow top). In Tg(CAG-LacZ/EGFP-KASH2) control littermates, no EGFP signal is observed and Nesprin2 is detected at the nuclear envelope of all Purkinje cells (white arrows bottom). <a href="https://www.jove.com/files/ftp_upload/53318/53318fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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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

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Behavior

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Genetics

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Immunology and Infection

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Neuroscience

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells

Source: Riedel, M. et al. <a href="https://www.jove.com/video/57525" target="_blank">Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration.</a> J. Vis. Exp. (2018)
This video describes a method for editing specific genes in prostate gland cells using an adenovirus-based delivery system. The approach allows for orthotopic and localized alteration of gene expression utilizing CRISPR technology to develop novel mouse models for prostate cancer.

Modelowanie raka prostaty w genetycznie modyfikowanych modelach mysich: technika zlokalizowanej edycji genów za pośrednictwem CRISPR/Cas9 w komórkach przedniego płata prostaty myszy

Source: Riedel, M. et al. Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration. J. Vis. Exp. (2018)
This video describes a method ...

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Establishing a Focal Demyelination Mouse Model via a Two-Point Injection of Lysophosphatidylcholine

Source: Pang, X., et al. <a href="https://app.jove.com/v/64059">A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice.</a> J. Vis. Exp. (2022)
This video demonstrates the generation of a focal demyelination mouse model using a two-point Lysophosphatidylcholine (LPC) injection. The skull of an anesthetized mouse is exposed. The injection coordinates are determined, and a hole is drilled to enable LPC injections at targeted sites. LPC induces focal demyelination of neurons via inflammation, while a two-point injection ensures its sustainability.

Source: Pang, X., et al. A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice. J. Vis. Exp. ...

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

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Przeglądaj więcej filmów

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Mouse In Vivo Placental Targeted CRISPR Manipulation

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Mouse Genome Engineering Using Designer Nucleases

Modelowanie raka prostaty w genetycznie modyfikowanych modelach mysich: technika zlokalizowanej edycji genów za pośrednictwem CRISPR/Cas9 w komórkach przedniego płata prostaty myszy

Establishing a Focal Demyelination Mouse Model via a Two-Point Injection of Lysophosphatidylcholine