The overall goal of this procedure is to alter gene expression in the neuron embryonic central nervous system. This is accomplished by first obtaining timed pregnant females and preparing them for surgery. The uterine horns are then exposed by generating an abdominal incision.
Once the embryos are exposed, DNA is injected into the lateral ventricle, third ventricle, or subretinal space. The final step of the procedure is to apply an electric current to the embryo through the uterine wall, orienting the paddle electrodes such that the positive pole is placed over the preferred site of transfection. Ultimately, results can be obtained that show the proliferation, migration, and differentiation of neuro progenitors in the tline kelon dying kelon and retina through immunofluorescence microscopy.
The main advantage of this technique or existing methods, like the degeneration of transgenic mice, is that it is rapid, allows for higher throughput studies and has a lower cost. This method can help answer key questions in the field of neural development, such as which genes regulate neural progenitor, cell proliferation, cell fate specification, neuronal differentiation, and cell migration. The implications of this technique may extend towards a better understanding of the genetic pathways that underlined defects in CNS development, many of which results in cognitive impairment and are behavioral disorders in humans Beyond its usefulness in understanding central nervous system development.
This technique can also be applied to other organ systems such as the developing peripheral nervous system, heart and placenta. It can also be applied to study disease models in mice as well as other model organisms such as chick and xip. Generally, the primary obstacle to success for individuals new to this technique is embryo Survival care must be taken while manipulating, injecting and poring the embryos.
We first had the idea for this method when we required a rapid means to manipulate gene expression in the embryonic, telencephalon and retina. We developed this technique in our laboratory by modifying the technique initially described by Sal, published in the journal Developmental Biology in 2001. We also worked with a crash lab to establish this protocol in the developing dilon as well.
The visual demonstration of this method is critical as the injection and electroporation steps are difficult to learn because they require a very careful manipulation of the embryos and the sites of injection at times can be very small. Example, the subretinal space Demonstrating the procedure today will be Rajiv, a postdoc from the Sherman's Laboratory To begin clean and sterilize all surgical tools. Next, fill syringes with saline solution and lactated ringer solution and place them on a heating pad to warm in the surgical area.
Sterilize a heating pad with 70%ethanol, and then cover it with a sterilized limp less gauze pad. Set the heating pad to 37 degrees Celsius. Place the animal in a small anesthetic chamber and anesthetized by delivering 5%isof fluorine with oxygen at one liter per minute through a vaporizer.
Being sure to collect the isof fluorine exhaust using a scavenging system. Once the animal's breathing is deep and regular, remove it from the chamber. Use toe pinching and eye blink reflexes.
To ensure full anesthesia. Place the anesthetized animal on its belly and use a respirator tube to deliver 2.25%ice of fluorine during surgery. Inject buprenorphine at a concentration of 0.1 milligrams per kilogram body weight subcutaneously at the nape of the neck.
Continue to monitor the animal's breathing and adjust the flow of isof fluorine if necessary. Then tape across the chest of the anesthetized animal and check again for a retraction response by pinching the hind foot with blunt forceps. Using a sterile cotton tip swab, spread a thick layer of de cream onto the abdomen ensuring that the under fur is completely coated.
Leave the cream on for approximately three minutes and check intermittently for hair removal. Using a sterile swab, wet a sterile gauze pad with sterile water and wipe the hair off the abdomen. Next, sterilize the incision site with chlorhexidine and more water.
Dry the abdomen with clean sterile gauze. Repeat this step with 70%ethanol using fresh sterilized cotton gauze. Prepare for surgery by donning surgical garb to expose the uterine horns.
First, locate the linear alba as the faint line under the skin at the midline Using forceps, pull the skin away from the abdominal wall and cut a small straight incision from the mid abdomen downwards with skin scissors using curved forceps and scissors. Repeat the process with the peritoneum. Grasp the peritoneum with the forceps.
Pull away and then cut an incision just large enough to pull the uterus through. Place sterile gauze with a small vertical slit over the incision and dampen it with warm sterile saline. Next, slide the forceps into the incision and locate the uterus holding the incision open with the forceps.
Grasp the uterus between the first and second visible embryos and pull both sides of the uterine horns onto the gauze. Count the number of embryos noting any resorption or embryos with a small body size. In order to prepare the correct number of DNA injections, wet the uterine horns with saline and carefully return one uterine horn into the abdomen.
To loath the surgical needles, use a pulled past up pipet with an attached mouthpiece to draw up 10 microliters of three milligrams per milliliter. Endotoxin free DNA solution containing methyl green dye and expel the DNA into a surgical needle. Mount the first filled needle onto the micro manipulator.
Stand attached to the injector starting with the embryo closest to the ovary. Use circular forceps to gently turn the embryo within the decidua so that it is positioned face forward, allowing injections to occur in a ROS roll to cordal direction. Using a fiber optic light source to identify the midline of the embryonic brain.
Note the flanked large lateral ventricles in rostral regions. Hold the embryo in place using curved forceps and position the filled surgical needle above the uterus such that the angle of entry into the brain is approximately 45 degrees. With a quick and steady motion, turn the dial on the micro manipulator to insert the needle tip through the utero wall and into the lateral ventricle of the embryonic brain.
Next, press the footpad of the micro injector. To inject the DNA solution, a successful injection is easily visualized by spreading of the methyl green dye throughout the embryonic brain ventricles. Now that the DNA has been injected, slowly draw the needle back up to remove it without causing breakage.
Place the injected embryo on the sterile gauze and wet thoroughly with warm saline. Grip the uterus with the positive pole of the paddle electrode placed over the desired site of transfection. Then deliver five square electric pulses of 40 to 50 volts and 50 milliseconds at a rate of one pulse per second.
Cover the injected and electro rated embryo with protective wet gauze, and then continue on to inject the next embryo. Upon completion of injecting and electro pering of all desired embryos in the uterine horn, carefully push it back into the body cavity. Remove the second uterine horn to begin injection and electroporation of the remaining embryos.
Once all embryos are back inside the uterus, add two to three milliliters of warm saline to the abdominal cavity, and then remove the gauze from the abdomen to reveal the sides of the peritoneum. Use black braided silk line to suture the peritoneum by anchoring the line on the peritoneum closest to the sternum and proceeding to stitch away from the anchor. Ensure all sutures are tight and that no underlying organs or skin are sewn into the peritoneum.
Clip the remaining suture line and discard. Pull the skin closed over the suture line with forceps using skin closure staples. Clip the skin taking care not to staple the suture line underneath or to pull the skin too tightly.
Once finished, ensure staples are tight by gently pressing together with the suture tool here, A-P-C-I-G two expression vector driving the expression of GFP has been electroporated into the developing neocortex at E 12. As neocortical development proceeds, the apical progenitors differentiate into either secondary basal progenitors or post mitotic neurons and continue to express GFP. These neural cells can then be analyzed for expression of typical markers of the neural subtype shown in red.
In this venal view of a forebrain of an embryo electroporated at E 12 and dissected at E 14, the electroporated site is indicated by the green epi fluorescence by E 14. GFP expression is seen in the thalamus, pre thalamus and hypothalamus indicating that dying lic territories have been successfully transfected. The dash box marks the location of section through one ventricular zone.
A closer look at the ventricular zone shows the migration of GFP cells outta the ventricular zone and into the mantle zone. Where hypothalamic nuclei will form the embryonic retina is also readily electroporated as shown in this coronal section through an E 16.5 I electroporated with PCIC at E 15.5. Here, the accumulation of m cherry positive cells is specific to the outer neuroblast layer and is not seen in B rrn three B positive retinal ganglion cells in the ganglion cell layer Once mastered, this technique can be done in 30 minutes if no unexpected complications arise.
While attempting this procedure, it's important to use proper sterile techniques, a steady hand when manipulating the embryos, and to have all of the necessary materials ready prior to putting the pregnant female under anesthetic. Following this procedure, electro braided embryos can be allowed to live until adulthood, at which point other methods like behavioral and functional assays can be performed to answer additional questions pertaining to how the manipulation of certain genes in the embryo affect neuronal function in the adult. Since its development, this technique has paved the way for researchers in the field of CNS development to explore gene function in vivo in mouse.
After watching this video, you should have a good understanding of how to introduce foreign DNA into the embryonic neural tube. Specifically, we show you how to prepare a pregnant female for surgery. Expose the uterus, inject DNA and electrolyte the embryo.
Don't forget that working with isof fluorine can be hazardous, and precautions such as a scavenging system should be taken while performing this procedure.
