This protocol allows us to perform a genetic manipulation in vivo in a key animal model that allows us to study the expansion and folding of a developing neocortex. The main advantage of this method is in using high spatial and temporal specificity in targeting those neural stem cells that are the key cell type for brain development. Before beginning the procedure, pull glass capillaries on a micro pipette puller and use forceps to cut off the distal part of the capillary to adjust the diameter of the capillary tip.
On embryonic day 33, add the appropriate concentration of DNA in PBS, supplemented with 0.1%Fast Green with gentle mixing, and place a 3-hour-fasted, anesthetized, pregnant female ferret on an operation table with a heat pad. Confirm the appropriate level of sedation by touching the periocular skin and pinching the skin between the second and third or third and fourth toes of both hind limbs. Isoflurane can cause adverse effects, including nausea and dizziness.
When handling isoflurane in liquid form, use the protective equipment. During the surgery, use appropriate canisters to capture the gas. Inject the animal subcutaneously with analgesic, antibiotic, and glucose, and place ointment on the animal's eyes.
Use clippers to shave the abdomen, and clean the exposed skin with a water, soap, and iodine solution. Then, dry the skin with gauze swabs and disinfect with alcohol and iodine scrubs. For in utero electroporation, place a sterile drape over the animal and use a scalpel to make an approximately 5-centimeter skin incision at the linea alba.
Use scissors to cut through the muscle layer and place gauze swabs around the incision site. Wet the gauze with PBS and place the uterus onto the swabs. Using a pipette with a long tip, load one of the pulled glass capillaries with 5 microliters of DNA solution per embryo to be injected.
Attach the loaded capillary to a holder and connect the other side of the holder to a tube and a mouthpiece. Locate the head of the first embryo and place a fiber optic light source next to the head. Using the pigmented iris as a reference point, penetrate the skin, skull, and cerebral tissue with the tip of the glass capillary and use mouth pipetting to facilitate the delivery of 3-5 microliters of the injection solution into the ventricle of one of the cerebral hemispheres.
Because the injected solution contains 0.1%Fast Green, a successful injection will result in a dark green staining of the injected ventricle, which will now be visible as a kidney-shaped structure. After the injection, place the tweezer electrodes on the uterus above the head of the embryo with the positive pole above the area to be targeted and the negative pole below the injected area. Set the pulse length of the electroporate to 50 milliseconds, the pulse voltage to 100 volts, the pulse interval to one second, and the number of pulses to five.
When all of the parameters have been set, press Pulse"and quickly drop several drops of warm PBS onto the electroporated embryo. When all of the embryos have been electroporated, return the uterus to the peritoneal cavity and use a 4-0 suture to close the muscle layer with the peritoneum. Close the skin in a similar manner and cover the wound with aluminum spray.
Then return the animal to its cage with a heat source and monitoring until full recumbency. Four days after electroporation at embryonic day 37, most of the targeted cells and their progeny are still in the germinal zones and the cells are seldom observed further basally within the cortical plate. The progenitor identity can be examined by immunofluoresence for markers of cycling cells, such as PCNA, whereas a subset of progenitors undergoing mitosis can be shown by markers such as phospho-histone 3.
At post-natal day zero, eight days after electroporation, the progeny of the targeted cells spread to all of the histological layers. Using a combination of transcription factor markers, different basal progenitor populations can be revealed. For example, Sox2 is a marker of proliferative progenitor cells, including basal radial glia.
And T-box brain protein 2 is a marker of neurogenic basal progenitors, which are mainly intermediate progenitors. By post-natal day 16, a majority of the targeted cells stop dividing and differentiate into neurons and glia, with ferret post-natal day 16 neocortex exhibiting the characteristic folding pattern. The in utero electroporation of ferret embryos is an extremely powerful method to study the function of genes.
It has allowed us to study genes that have been implicated in the expansion of the neocortex in evolution, including human-specific genes. And using this powerful method, we have indeed been able to find out key features of how the evolutionary expansion of the human neocortex likely worked.
