The overall goal of this procedure is to select successfully in utero electroporated wrap-ups for further analysis. This is accomplished by first injecting the target DNA and then performing an in utero electroporation for the selected area within the brain. The electroporated embryos develop within the mother's uterus and are born normally live.
2D imaging is used to identify successfully electroporated animals at defined postnatal time points. Finally, the targeted area is characterized in more detail by 3D imaging. Ultimately, these topically transgenic rats can be used in behavioral testing to show the consequences of disturbed neurodevelopment on adult behavior.
While a electroporation is an established technique, it has not been widely used for the generation of topically transgenic adult animals. This is in part due to the technical skills required, but also to our present inability to select only for the successful electroporated newborn pups. We present a solution to this problem by using live in vivo bioluminescence imaging To allow successful in vivo bios essence imaging of a neutral electro rats.
Lucifer rays reported gene is co electroporated. Luminescence detection can be activated by Luciferian injection to monitor the approximate position of the rated cells. This then allows to select for successfully electroporated red pops.
To begin collect together the sterile materials needed for in utero electroporation. Next, prepare the DNA solution that contains the target D-N-A-G-F-P vector, luciferase vector, and fast green dye. Under ISO fluorine anesthesia expose the uterus to visualize the embryo to be injected.
Using a thin glass needle, inject the DNA solution into one of the lateral ventricles of the embryo. To hit a cell population of upper cortical layers, place a seven millimeter electrode around the head of the embryo and place the positive electrode above the ventricle with a slight dorsal lateral tendency. Perform the electroporation with a square wave pulse electroporation by stepping on the foot pedal to target hippocampal cells.
Place the positive electrode on the opposite side of the injected ventricle. Before electroporated first dilute Lucifer sodium salt in PBS to a concentration of 10 milligrams per milliliter and sterilized by sterile filtration. After weighing hold a pup with the abdomen up and slightly stretched before injecting the Lucifer to begin the imaging procedure.
Lucifer injected pups are anesthetized and placed in a prone position within the Ivis spectrum device. First 2D bioluminescence images are required to determine which pups were successfully electroporated working in the Ivis acquisition control panel, select the bioluminescent imaging mode to take overhead photos at the beginning of the measurement. Check the photograph setting and set the binning to medium and the F-stop to eight select Use subject height under the focus setting.
Next, set the excitation filter to block the emission filter to open and the binning to medium. The F-stop should be set to one and the stage level or field of view is set to a acquire A 2D image. With luminescence exposure time of 180 seconds, the electroporated areas of positive pups are then investigated further by taking 3D images following settings for firefly luciferase.
First, confirm that the photograph option is selected to acquire an overhead photo at the start of imaging. To scan the surface of the animal prior to bioluminescence measurement, select the structure option for pups up to two weeks of age. The following emission filters and exposure times are selected due to the decrease in signal strength in older animals, the exposure time is increased for the three best emission filters in rats older than P 20.
Next, set the stage level to be the benning to medium and the F stop to one and acquire the 3D image. After imaging mark the rats with distinct ear holes to differentiate them from each other and to match them to the Ivis live imaging pictures. Finally, remove the animal to a warmed surface until fully recovered.
3D images are generated using the living image software pre-installed on the Ivis spectrum system. The first step is to reconstruct the surface topography. To do this crop a region of interest and set the threshold between 20 and 30%Start DLIT 3D reconstruction for brain tissue with an image threshold of 10%for each wavelength setting.
To create 3D movies, select the animate button Within the 3D toolbar, manipulate the orientation and zoom of the 3D image as desired. Next, press the record button to track the changeover from one position or orientation to another. In these examples, P seven rats imaged after a luciferian injection showed either no luminescence signal, a weak signal, or a strong luminescence signal depending on the electroporation success.
These images show a timeline of consecutive bioluminescence measurements in the same rat demonstrating a long detection window. Here 2D images show successful electroporation into the cortex or hippocampus. The targeted areas are further visualized using 3D imaging in both cortically and hippocampal electroporated animals.
This video shows the successfully electroporated area from multiple perspectives. In this example, the targeted locations identified from 2D and 3D imaging in the live animal are validated further through bioluminescence and GFP epi fluorescence detection in the dissected brain. This image taken from the same successfully electroporated rat brain shows a detailed view of the GFP expressing cells at P 14 When performed correctly.
This method allows for the study of the behavioral and neuropathological consequences of neurodevelopmental defects. Selecting only the successfully electro rated rats greatly reduces costs and effort. Success with this technique requires practice and has variable motility and efficacy rates.
Immuno histology of core electro GFP is important to document the exact area of transfection at anatomical resolutions for post-hoc correction of behavioral results.
