The overall goal of this procedure is to electro operate charged macro molecules into cranial facial mesenchyme. This is accomplished by first positioning an anesthetized stage 28 embryo within the electroporation chamber. The next step is to micro inject the solution containing charged macromolecules into the C craniofacial mesenchyme.
This is quickly followed by application of an electric current, thereby introducing the charge nucleotides into cells else. The final step of the procedure is to visualize the electroporated tissue by fluorescence microscopy. Ultimately, results can be obtained showing changes in cranial facial tissues.
These results can be analyzed in vivo or in fixed tissue using fluorescence, microscopy, or immunohistochemistry. The main advantage of electroporation over existing methods like microinjection and mutation are tissue and temporal control of our molecular tools, demonstrating our procedure today will be Jackie Tabler, a postdoc in my lab. The pair of L-Shape electrodes required for this procedure is made from high purity, 0.4 millimeter tungston wire for each electrode first cut eight centimeters of the wire.
Then using putty such as blue tack affects a one milliliter syringe at the midpoint of the tungsten wire, leaving four centimeters of wire exposed from the tip of the syringe. Bend the tip into an L shape one centimeter from the end. Trim the tip so that the end measures 0.5 centimeters in length.
This tip is the electrode terminus run the excess tungsten wire parallel to the syringe. It'll be used to connect the electrode pulse generator after the second electrode has been made. Fasten the pair of electrodes to each other using tape so that the electrode terminal I run in parallel, one centimeter apart.
Finally, attach the electrodes to a square wave pulse generator via DC cables. To prepare the custom made electroporation chamber, line the bottom of a 90 millimeter dish with around five millimeters of non-toxic plaster scene modeling clay. Fill the dish with electroporation media immersing the plaster scene.
Then use number five watchmaker's forceps to carve a T-shaped well in the plaster scene to form the electroporation. Well, the long side of the well should measure around two millimeters by two millimeters by 10 millimeters, and the short side should be two millimeters by two millimeters by five millimeters. Micro pipettes for this procedure are made from silicate glass capillaries.
Use a needle puller to prepare micropipets with an eight to 12 millimeter long taper and a fine tip, crush the tip around two millimeters from the tip using forceps, creating a jagged break to set up the micro pipette. First, fill it with around one microliter of injection solution, which in this demonstration contains fluorescently labeled oligonucleotides. Next, secure the micro pipet in a micro manipulator angle the micro pipette at 50 degrees from the tabletop.
Set the injection pressure at 20 PSI and calibrate the micropipet to inject 30 liters per pulse. In preparation for electroporation, anesthetize a stage 28 xeno larvae by incubating it for five minutes. In electroporation media, which is normal amphibian media containing 0.1%Benzocaine transfer the anesthetized tadpole into the electroporation chamber.
That is filled with electroporation media. The trickiest bit of the procedure is inserting the micro pet into the tadpole. So to ensure success, you have to secure the embryo really tightly and then quickly apply the current after the micro injection.
Position the embryo within the long well so that the head rests in the T junction. With its dorsal side down and ventral side exposed, the head should be slightly elevated compared to the tail using forceps, gently secure the tadpole in the well with the surrounding plaster scene. It is important to secure the tadpole to prevent it from twitching during electroporation, which could result in severe damage to facial tissues.
To begin this procedure, insert the micro pipette tip immediately posterior to the cement gland and into the facial mechy. Inject 30 nanoliters of solution into the mesenchyme, retract the micro pipette. Quickly align the electrode tips parallel to the head of the embryo, and apply eight 50 millisecond 20 millivolt square pulses.
Then retract the electrodes using forceps. Carefully release the tadpole from the well. Then use a pipette to transfer the tadpole to threequarter nom with 0.025 milligrams per milliliter.
Gentamycin tadpoles can be incubated in this media overnight or longer after 24 hours. Screen tadpoles by fluorescent microscopy for efficient electroporation. The use of fluorescent molecules allows easy screening of electroporated embryos.
This figure shows a typical batch of morph oligonucleotide electroporated tadpoles around 1248 and 96 hours after electroporation. At stages 30, 34 and 44 respectively. Fluorescent morph oligonucleotides can be visualized within the cranial facial meen at stages 30 and 34.
At stage 44 fluorescent can be detected in cartilages indicated by the white arrow heads. The highly autofluorescent area is the gut. Following this procedure, we can track the lineage of electro cells as well as the molecular requirements for proteins of interest.
This approach can be combined with live imaging techniques to study gene function over time during craniofacial development.
