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

The overall goal of this experiment is to illustrate the usefulness of a transgenic mouse model, allowing for the inducible and targeted expression of a fluorescently tagged EGFP cache, two peptide in vivo, and the resulting disruption of endogenous link complexes. In this experiment expression of fluorescently tagged cache two will be targeted to cerebellar Perkin cells. This genetic approach can address key questions about the role of link complexes in nuclear positioning within different most tissues.

The inducible and targeted expression of E-G-F-P-K two has several advantages. First, it requires a single breeding scheme of cay mice with any existing cruise strain. Second, this approach bypasses the perinatal ality observed in mouse models of germline mutations of link complex components.

Finally, this method allows to study the physiology of link complexes within specific cells or tissues. In vivo. Chloe Potter, a member of the lab, will show you the difference steps required to visualize E-G-F-P-K two in cerebellar Burkin G cells.

To begin breed clac Z mice with PCP two CRE mice to produce PCP two CAG C two mice as described in previous publications. When the resulting pups are born, perform genotyping. To identify those harboring both EGFP and Cree Transgenes as previously described, prepare 50 milliliters of a 30%sucrose solution in PBS and store it at negative 20 degrees Celsius until the day of tissue harvesting at the appropriate age, perform a trans cardio perfusion of the mice under anesthesia in order to prepare the brain tissue for dissection as described in the written protocol, place the dissected brain in a 10 centimeter tissue culture plate with 10 milliliters of PBS and separate the cerebellum from the cerebra.

Next, use a scalpel to dissect the cerebellum in the sagittal plane and transfer the sections into 30%sucrose solution and store it at four degrees Celsius overnight. Prepare a slurry of dry ice and two methyl butane. Allow the temperature of the bath to equilibrate for five minutes.

Next, fill a cryo mold with OCT compound and place one half of the dissected cerebellum into the mold using a dissecting microscope and a pair of forceps. Position the cerebellum so that the bi dissected surface that is near the midline of the cerebellum is facing up in the mold. Then transfer the cryo mold into the dry eye slurry for five minutes until the OCT compound has formed a solid white matrix around the tissue specimen.

Store the embedded specimen at negative 20 degrees Celsius until needed. Label pretreated polylysine slides for collecting tissue sections and mount the block of cerebellar tissues on the tissue holder with fresh OCT. Once the block is firmly attached, mount the tissue holder and shave 40 micrometer slices to remove excess OCT until the tissue is reached.

Set the thickness at 15 micrometers and collect the sections by carefully contacting the OCT section with The polylysine coated side of the slide. Store the tissue sections at negative 20 degrees Celsius until proceeding with staining and imaging. When ready to continue, thaw the cerebellar sections in the dark to prevent fading of the fluorescent proteins.

Using a hydrophobic barrier pen, draw a rectangle around the perimeter of the tissue sections. Add about 500 microliters of 4%paraform aldehyde to the sections for five minutes to postfix the tissue. Then use a vacuum to aspirate the fixative being particularly careful not to disturb the tissue sections with a pipette.

Gently apply PBS on the slide without disturbing the tissue sections. After five minutes, use the vacuum more pipette to carefully remove the liquid, repeat the PBS rinse a total of three times. Next, add about 500 microliters of blocking solution to the slides and incubate them for 10 minutes.

Remove and then replace the blocking solution with the same solution containing one to 300 dilutions of either cal binding or nesin two antibodies. Then remove the antibody solution and rinse the slides with PB S3 times. Incubate the slides for one hour at room temperature in the dark.

Next, prepare one to 1000 dilution of anti-US Alexa floor 5 94 for the cow binding labeled slides and anti Alexa Fluor 5 94 for the Nesprin two labeled slides in the blocking solution and store the solutions in the dark until they are needed. Gently remove the final PBS rinse and cover the sections with the diluted secondary antibodies for one hour in the dark. Following the incubation, remove the secondary antibody solution.

Then apply nuclear stain such as DPI at 300 nanomolar in PBS for five minutes. Afterwards, rinse the sections twice more in PBS. Remove as much PBS solution as possible and cover the tissue section with one drop of mounting medium.

Place a glass cover slip on top of the tissue section avoiding air bubbles and set aside for 15 to 30 minutes at room temperature in the dark. Then use a fluorescent microscope to visualize the antibody staining and fluorescent proteins. For cerebellar slices, use either a 20 x or 40 x objective.

To view the E-G-F-P-C two nuclear rims as shown here, E-G-F-P-C two can be directly visualized in cerebellar slices in the Fitzy channel. Furthermore, the colocalization of EGFP with cow binding, which specifically labels Perkin cells, confirms that E-G-F-P-C two expression is confined to perkin cells within Perkin cells. EGFP cache two is targeted to the nuclear envelope as indicated by EGFP positive RIM like patterns observed around the nucleus.

In this example, EGFP cache two expression was detected in more than 70%of cow binding positive Perkin cells. EGFP cache two displaces endogenous nesprin from the nuclear envelope through the saturation of available sun domains. Accordingly, endogenous NESIN two is displaced from the nuclear envelope of Perkin cells expressing EGFP cache two.

While Perkin cells that do not express E-G-F-P-C two retain endogenous nesin two at the nuclear envelope as expected control interate, which do not express CRE recombinase display NESIN two at the nuclear envelope of the whole bikini cell population One. So CT blocks of tissues are available. E-G-F-P-K two can be visualized in about four hours.

While attempting this procedure, it is important to remember that the extent of PFF fixation and or embedding conditions can drastically affect the antigenicity of your epitope. It is therefore very important to determine the optimal fixation conditions that preserve your specific epitope. After watching this video, you should have a good understanding of how to use the CK two transgenic mouse model to target the expression of VGFP cashew to specific tissues.