Using this method can allow for the discovery of how certain gene expression changes can attribute to pathologies associated with neurological diseases. The advantage of this technique is that it bypasses the need to create or purchase genetic animal lines which can take years to create and validate. This technique allows for the study of genes implicated in neurological disorders.
Determining how specific genes contribute to pathology is an important step in developing novel therapies. The advantage of this protocol is learning to manipulate the embryos so that the experimenter accomplishes the gene transfer step without decreasing the survival rates. Visual demonstration of this method is important because the embryos are very fragile so it is helpful to observe how an experienced experimenter manipulates them during the procedure.
Bring pregnant dams to the surgery area at least 30 minutes prior to the surgery. Sterilize the entire surgery site using sterilized germicidal wipes and then 70%ethanol. Then sterilize autoclave tools in a glass bead sterilizer.
Transfer sterile PBS to 50 milliliter conical tubes and place the tubes in a water bath heated to 38 to 40 degrees Celsius. Check the sterile saline temperature with a thermometer. Turn on the water heating circulation pump so that it is warmed to 37 degrees Celsius.
Then turn on the pressure injector and electroporator and ensure proper function. Briefly spin the plasma DNA solution on a tabletop centrifuge and place it on ice. Pull glass pipettes on a pipette puller so that the tip of the pulled glass pipette is about 50 micrometers in diameter.
Then fill the pipette with 20 to 40 microliters of DNA solution. Set up all necessary items for surgery including hair removing lotion, iodine, 70%ethanol, cotton swabs, eye drops, sutures, and gauze. Prepare a surgery sheet and fill out the necessary information.
Weigh the mouse prior to surgery and record the weight. After anesthetizing the mouse, administer preoperative analgesics and remove the fur from the abdomen using hair removal cream or a razor. Sterilize the abdomen by swabbing it with povidone iodine and 70%ethanol three times.
Create a sterile field around the abdomen with sterile gauze and draping. Make a midline incision in the abdominal skin while lifting the skin up with forceps to avoid cutting through the muscle. Then lift the muscle up and cut through it taking care to avoid cutting vital organs.
Carefully pull the uterine horns out of the dam using ringed forceps and place them gently onto the sterile field making sure that they are supported with padding and aren't tugging too far away from the dam. Keep the uterine horn moistened throughout the rest of the surgery with the prewarmed sterile PBS. Position an embryo with either forceps or fingers, then carefully insert the pulled glass pipette into the lateral vertical and inject two to three microliters into each ventricle by inserting the pipette into one and then the other ventricle.
The ventricle has been successfully targeted if a crescent shape is present after injection. To transfect cells bilaterally in the frontal cortex, position the two negative electrodes on the sides of the embryo's head just lateral and slightly caudal to the lateral ventricles and position the positive electrode between the eyes just in front of the developing snout. Ensure the embryo is generously moistened and apply four square pulses.
Inject and electroporate all embryos, going one by one so that each embryo is electroporated immediately after the DNA solution injection. Once all embryos have been electroporated, carefully insert the uterine horns back into the abdominal cavity. Coat the abdominal cavity in sterile PBS to aid uterine horn placement.
Fill the abdominal cavity with sterile PBS so that no air pockets remain after suturing is complete. Suture the muscle with absorbable sutures and the skin with silk non-absorbable sutures. Allow the dam to fully recover in a heated chamber for at least one hour.
Check on the dams regularly in the next 48 hours. As the dam recovers from the anesthesia and regains consciousness, it will start moving and whisking. This method was used to transfect approximately 5, 000 layer 2/3 pyramidal neurons with the pCAG-GFP plasmid.
Most of these cells were localized to the frontal cortical regions. A representative example shows the rostral caudal distribution of transfected neurons in an adult control mouse, confirming the ability of bilateral IUEs to target and genetically label large populations of pyramidal neurons in the frontal cortex. The first part of the maternal interaction task tests the ability of control P18 transfected mice to find nest bedding.
Control mice spent more time exploring their nest bedding than exploring fresh bedding, suggesting that they have intact sensory motor abilities and exploratory behavior. The second part of the task takes advantage of the tendency of mice to be motivated to interact with their mother. In this task, pups spent most of their time near their mother while spending significantly less time exploring the empty cup or nest bedding.
Adult control mice spent approximately 35%of the time exploring a novel object. When presented with a novel and a familiar object, adult mice spent more time exploring the novel object. In the sociability task, control adult mice spent similar amounts of time exploring a novel mouse and an empty cup.
This behavior was automatically tracked with DeepLabCut software by labeling various points on a mouse, including the limbs, centroid, head, and ears. The software was also used to determine when mice were rearing by examining the length of the mouse's body since this distance becomes shorter when the mouse rears. Following this procedure, many cellular molecular questions can be answered by examining the tissue and assaying different measures such as spine density or expression levels of various proteins.
