After getting a sizable portion of ablated embryos fixed in ethanol at the necessary stages (st. 18 and st. 30), in situ hybridization is necessary to determine the gene expression patterns in the embryos. This procedure works by linearizing a large amount of plasmid DNA grown from E. coli cultures. E. coli is used to gather a large amount of DNA for use because they reproduce quickly. After linearizing the DNA, transcription is done in vitro to create antisense RNA that will bind to the location of the RNA for the gene of interest. This RNA is then labeled with fluorescent or colored probes so that the signal can be observed. The genes of interest used in this experiment are XCG-1, Otx2, En2, and Krox20. These are various neural marker genes that are expressed early and later in development. A control group of embryos also undergoes the hybridization procedure as a control to show where normal expression occurs in these embryo stages.
In these past few weeks, I have worked on practicing and perfecting my ablation procedure. For the procedure, a sharp needle and forceps are used to remove the part of the ectoderm that corresponds to portions of the brain and spinal cord. To do this, a special salt solution must be used that contains Ficoll, as using the standard salt solution does not allow the embryos to heal. If the embryos do not heal, then an in situ hybridization procedure cannot be performed to analyze the gene expression of the embryos. This is a very delicate procedure, as removing too much tissue will likely cause the embryo to lyse and removing too little tissue or incorrect tissue will produce bad data. After a good amount of practice, I would say that I can comfortably perform ablations accurately. After the ablation is performed, the embryos are placed under a microscope that takes pictures at certain intervals to display the wound healing capability of the embryos. The specific time interval that I use is 30 minutes with 30 second intervals (60 total images that can be played back in succession to show wound healing), but members in this lab in the past have taken images for up to two hours (where the original ablation is barely noticeable at that point). After the ablations, the embryos are grown up to desired stages (stages 18 and 30 in this experiment; ablations are performed at stage 11.5) and fixed in ethanol to stop the embryo from developing further. This allows gene expression to be analyzed through in situ hybridization. After getting a sufficient amount of ablated and fixed embryos to work with, in situ hybridization will be done using marker genes to show the recovery of the neural tissue post-ablation.
This first week in lab was really just learning how lab works. Although I started research here in the spring semester, I did not have time to do much as the upperclassmen were busy working on their own projects and did not have the time to show me around the lab. I have learned a lot about how this lab works and feel like I am ready to contribute. I learned frog care procedures and learned how the frogs mate and the mating rotations that are used in this lab to ensure that there are always embryos available for experimentation. I also became more acquainted with the process of collecting the embryos from the mating tank and moving them into the lab. I learned a bit about this last semester, but I never had the opportunity to do it by myself due to class conflicts interfering with the mating collection times. I also learned why each step for the collection procedure is done, as before I only knew to “do the steps” and did not really understand the science behind what I was doing. I also had more time to learn about sorting the bad embryos from the good embryos. If the bad embryos are left in the same plates as good embryos, the bad embryos will die and this will release signals to the other embryos in the plate. If the plate is left unattended for a long period of time, eventually all of the embryos will die due to this signaling. Therefore, sorting bad embryos is important after collecting each batch. The bad embryos are embryos that are undeveloped (meaning that they are only in the “1-cell” stage) or are malformed. I also learned how to make the salt solution that the living embryos are stored in. It is important to keep the embryos in this solution as the salt in the solution keeps the membranes from breaking, which would kill the embryos and ruin the batch. The solution also contains an antibiotic which keeps the embryos protected from bacteria since their protective jelly coating is removed during the collection process. Soon, I will hopefully learn the ablation procedure and run in situ hybridization tests on embryos to determine their gene expression patterns.
The Effect of Ablations and Perturbations on Gene Expression and Calcium Ion Activity in Xenopus Laevis
This project, “The effect of Ablations and Perturbations on Gene Expression and Calcium Ion Activity in Xenopus Laevis“ will show if there are any differences in gene expression and calcium ion activity when specific embryos in certain developmental stages and what these differences are and how they might affect further development. Ablation refers to cell ablation, which is the act of destroying cells in a given organism. Perturbation refers to temporary changes in environmental conditions that create large changes. Xenopus laevis (African Clawed-Frog) is used as a model organism because their embryos are very large compared to other vertebrates, making it easy to see the reactions to the conditions. Copious amounts of embryos are produced during Xenopus matings to allow for the necessary variation to get sufficient data. Ablations and pertubations will be conducted while new embryos are collected for mating. The embryos will then be allowed to grow to the desired developmental stage before development is halted. In situ hybridization will be used to determine if the disturbances affected gene expression.