The overall goal of this procedure is to knock down endogenous gene expression by injecting drosophila embryos with double stranded RNA to identify genes required for organ development. This is accomplished by first collecting and lining up embryos in a straight line. Next, needles are prepared and used to inject double stranded RNA into embryos.
Finally, embryos are placed into a moist chamber to develop. Ultimately, results can be obtained that show defects in drosophila tracheal branch fusion through antibody staining of late stage embryos, or by observing larvae under a brightfield microscope. The main advantage over this technique over existing methods like forward genetic screen to identify genes required for organ development is that this is a relatively simple and a quick reverse genetic approach to analyze gene functions before a mutant is available for that gene.
This method can help answer key questions in the developmental biology field, such as the identification of critical genes required for the development of the D tracheal system. This is an excellent model to study the morphogenesis of mammalian organs such as the vertebrate airway, circulatory system, kidney ducts, and exploratory epithelial. Generally, individuals new to this method will struggle because it takes time to learn the methods such as lining up embryos, breaking needles, and injecting stranded RNA into the embryos.
Visual demonstration of this method is critical as the steps involving the amount of double stranded. RNA injected varies between embryos and written demonstration of what syncytial, blasto derm embryos look like can be difficult. These steps are difficult to learn because injecting suitable amounts of double stranded RNA into syncytial blasto embryos is such a critical step to effectively knocking down the expression of endogeneous genes.
To begin set up cages at 25 degrees Celsius using two to four day old W 1 1 1 8 flies change the grape juice plates every hour during the day to synchronize the egg collection over a one to two day period. Before the collection. Collect syn city or blaster derm embryos for one hour at 25 degrees Celsius, a rectangular piece of grape juice agar then lightly cut a line in the middle of the agar with the razor blade.
Use a metal probe to transfer embryos from the grape juice plate to the piece of grape juice.Agar. Line up around 50 embryos in a straight line with the embryos, longer axes at a 45 degree angle to the line in the middle of the agar. Ensure that all the embryos point in the same direction with their posterior ends pointing away from the line.
Cut a rectangular piece of double-sided tape and stick it on an 18 by 18 millimeter cover slip. Pick up embryos using the tape on the cover slip. Place the cover slip on a glass slide.
Press the embryos slightly to the double-sided tape and allow the embryos to air dry for about 10 minutes. Cover the embryos with a thin layer of 700 halo carbon oil. Wait for around 10 minutes until the embryos are clear under the microscope.
Needles are prepared for microinjection by pulling glass capillary tubes. We use a needle puller from World Precision Instruments under the program settings. Heat one delay four back.
Load the needle with two microliters of double stranded RNA using an Einor P 20 pipette with einor micro loader tips. Prepare a slide with a clean cover slip on top of it and tape them together. Bring the filled needle against the edge of the cover slip, creating a sharp point.
Double stranded RNA leaks out of the needle tip. When the foot pedal of the pico pump is depressed, prepare a slide with a drop of oil in the middle. Insert the needle into the oil drop.
Adjust the setting on the pico pump to expel 100 to 200 picoliters of double stranded RNA when the foot pedal is depressed. Double stranded. RNA can be seen as a small droplet position.
The needle tip at the posterior portion of the first embryo depress the pico pump foot pedal. Double stranded RNA will appear as a small. in the injection site.
Finish injecting double stranded RNA into the embryos on the slide. Kill all non blaster derm embryos. Place the cover slip into a covered moist chamber, such as a plastic Petri dish at 18 degrees Celsius until the embryos developed to the desired embryonic or larval stage.
For embryos that have developed to the desired embryonic stage, use a probe to remove the embryos from the double-sided tape and push them to the edge of the cover slip. Wash the embryos off with heptane into a collection vial with nylon mesh at the bottom. Wash the embryos three times with PB tating in 50%Bleach for five minutes and wash the embryos again three times with PBT.
Transfer the embryos from the collection vial to a piece of nylon mesh using a pipette dip the nylon mesh piece into the fixative solution. Fix the embryos for 30 minutes. Remove the lower layer of the fixative solution.
Then ize them with methanol embryos are transferred to an eppendorf tube and immunostain with two A 12 and diffusion antibodies using standard antibody staining protocols for embryos that have developed to the desired larval stage. Prepare a slide with a drop of halo carbon, 700 oil in the middle, and two small cover slips on each side of the oil drop. Transfer one larvae to the slide and then apply a cover slip on top of the oil drop.
Observe the larvae under a brightfield microscope. Representative results of both diffusion double stranded RNA and GFP double stranded. RNA injection are shown here.
GFP double stranded. RNA injected embryos show normal tracheal fusion and diffusion protein is present in fusion cells. Arrows point to lateral trunk diffusion positive fusion cells and the asterisk indicates the location of normal fusion.
However, diffusion double stranded RNA injected embryos show failed branch fusion and diffusion proteins are not present in fusion cells. The triangle indicates the site of failed branch fusion when double stranded RNA injected embryos developed to the larval stage. The G-F-P-R-N AI larvae show normal tracheal branch fusion indicated by asterisk.
On the other hand, the diffusion RN AI larvae demonstrated failed branch fusion indicated by triangles. These results show the effect of knockdown of endogenous diffusion gene expression by double stranded RNA injection into drosophila embryos. While attempting this procedure, it's important to remember to kill all enseal blaster embryos After its development.
This technique paved the way for researchers in the field of developmental biology to explore the function of novel genes for organ development in drosophila. After watching this video, you should have a good understanding of how to knock down endogenous genes by injecting double stranded RNA into drosophila embryos.
