Our goal is to elucidate the physiological roles of genes using mice as an experimental model. We also focus on developing mouse models of specific diseases that scientists can use to investigate disease causation and develop effective treatments. An example of such activity is the mouse model of Angelman syndrome.
Arguably, the most promising new technology with potential is integrating artificial intelligence based on deep learning techniques in animal research. This approach can be used to monitor animal behavior in the home cage directly without the need for the experimenter to disturb the animals. The biggest challenge in behavioral studies currently is reproducibility.
Therefore, we introduce a battery of tests that validate the Angelman syndrome mouse model in a way that does not depend on the model's age or species. We use established behavioral tests to validate our AS model, however, with the novel approach for the model development closely resembling the human condition with a large deletion. Using CRISPR technology, we deleted the entire UBE3A gene from the mouse genome in contrast to other models where the deletion is limited to 3KB.
We are working on developing a genetic therapy that could restore any symptoms in a mouse model. To begin, place the plus-shaped maze on the testing platform just below the camera. Using the potentiometer on the wall, adjust the light intensity to 70 lux at its center with the help of a luxometer with its sensor placed at the center of the maze during the adjustment.
Open the software by double-clicking on the viewer software icon and load the configuration for elevated plus maze or EPM testing by clicking on the icon on the upper left side of the configuration tab. Enter the animal information into the corresponding fields of the experiment tab, including animal ID, genotype, gender, and experiment information. Verify that the zone's position, open arms, and closed arms are correctly configured.
Using a visual control and computer mouse, ensure that the virtual outline zones match the corresponding EPM zones. Place the mouse cursor on the arrow icon on the upper left side of the acquisition tab. Remove the animal from the home cage and place it gently in the center of the EPM.
Start the experiment by left-clicking on the computer mouse. After five minutes, save the recorded data by clicking OK.Name the file and click Save. Export the results to CSV file for offline analysis by clicking on the icon on the left vertical panel of the data analysis tab.
In the EPM test, none of the parameters related to anxiety-like behavior were altered in maternal heterozygous UBE3A mutants. Place the four open field or OF test boxes on the testing platform below the camera. Using the potentiometer on the wall, adjust the light intensity to 200 lux at the center of each OF test with the help of a luxometer, with its sensor placed at the center of each box during the adjustment.
Open the software by double-clicking on the viewer software icon and load the configuration for OF testing by clicking on the icon on the upper left side of the configuration tab. Enter the animal information into the corresponding fields of the experiment tab. Verify that the zone's position matches the OF test boxes and adjust them if needed.
Using a visual control and computer mouse, ensure that the virtual outline center and periphery zones match the corresponding OF test zones. Place the mouse cursor on the arrow icon on the upper left side of the acquisition tab. Remove four animals from the home cage and place them in the corner of each test box.
Start the experiment by left-clicking on the computer mouse. In the OF test, the maternal heterozygous UBE3A mutant animals were significantly hypoactive as reflected by a shorter traversed distance, lower average speed, and longer resting time. However, none of the parameters related to anxiety-like behavior were altered in UBE3A mutants.
Connect the tail suspension cage to the computer using a USB cable. Insert the USB dongle into the computer and start the software by double-clicking on the BIO TST software icon. In the settings tab under Global, ensure the acquisition duration is set to 360 seconds.
In the Experiment tab, select the new list of subjects and create a new experiment file and a new list of tested subjects by following the instructions in the opened tab. Prepare the animals for the test by wrapping single-sided adhesive tape around 3/4 of the animal's tail starting from the base. Start the run by clicking Start Run.
Then continue in the acquisition tab. Immediately after hanging the animal on the hook, click the start icon under the visualized position to acquire data. After testing all the animals, in the analysis tab, select the last four minutes of the acquisition for analysis.
Select all valid runs in the analysis period and click Analyze Selected Subjects. Choose the desired data format and click Export Selected Data to export the collected data. In the tail suspension test, UBE3A mutant animals were immobile for significantly longer than their control littermates, indicating their depression-like behavior.
Turn on the treadmill and manually set the belt speed to 20 centimeters per second on the equipment panel before switching on the apparatus slide. Launch the DigiGait Imager software and set the shutter speed to 100 for albino mice or 130 for black or dark mice in the field for shutter speed. Place the mouse onto the treadmill belt and confirm that the mouse can perform the test by setting it to a slow walking speed for around three seconds and then stopping it while observing the mouse continuously.
Start the belt by pressing the start button and record for 10 seconds. Ensure that a clear and fluent locomotion of at least 10 to 15 steps is observable. Stop the belt by pressing the stop button and return the mouse to the temporary holding cage.
Screen the recording for a sequence of images with fluent steps by clicking play and reviewing the recording with the visual control in edit mode. Select 10 to 15 fluent movements by manually writing their starting and ending frame numbers into the relevant fields. Enter the animal information, including animal ID, date of birth, sex, weight, belt speed, and belt angle.
Save the file by clicking SAVE. Next, open the DigiGait Analysis software and perform gait analysis based on a fully automated analysis of video recordings of animal footprints. The gait indices of UBE3A mutant animals were altered.
UBE3A mutant animals had a longer swing and stance that resulted in prolonged stride duration and length. Mice's hind limbs'propulsion, duration, and deceleration were also increased. These findings also revealed a larger paw area at the peak stance.
Turn on the rotarod equipment and launch the software by double-clicking the rod software icon. Create a new file in the file tab and save it with an appropriate name. In the setup window, fill out the experiment details such as the date, user's name, and any eventual comments.
Set the speed profile to 300 seconds, initial speed to four RPM, and terminal speed to 40 RPM. Open the measuring panel by clicking Measure. Start the initial rotation of the rod at four RPM by clicking Start and place the first five animals in their assigned positions.
Start the testing protocol by clicking Start Profile, and the rod will gradually accelerate to 40 RPM over five minutes. After all the animals have fallen from the rod, close the measure window by clicking Close and click Show to display the collected data. Export the acquired data in CSV format by clicking Export CSV.
Rotarod performance showed a reduced latency to fall in UBE3A mutant animals. Take a cotton nestlet with forceps and record its weight manually using scales. Place the nestlet randomly in a cage but on the opposite side of the water supply.
Weigh each nestlet for the next four days manually using scales. Ensure that the nestlet is dry when weighed. If not, dry on a heating pad.
If the nestlet is torn into several parts, weigh the largest part. Record the weights on paper or in a pre-made spreadsheet. The UBE3A mutant animals used significantly less material to build their nests, and this difference was particularly prominent between transgenic females and their control counterparts.
