Welcome to the week 6 blog post! Because the aPKC probe I was originally planning to use didn’t work, I have been working my way through a few other probes. I need to use a probe that allows me to see the structure of the cell and the cleavage furrow. On Monday, I tried two different kinds of myosin probes. Interestingly one of these probes was formed by using antibodies from camels! Although one of the probes was visible, it wasn’t particularly bright or very helpful. So, on Tuesday we tried a utrophin probe. The probe has been used before in the lab so we knew it would work. The probe lets us see the cleavage furrow, and if the spindle poles are close to the surface, we can see them too. We have been trying to get videos of the embryo dividing during first cleavage, but the success rate is very low right now. Let me rephrase that. We have plenty of videos of the embryos dividing, you just can't see the cleavage furrow because the embryo rotated around to hide the division! If I can see the spindle poles I can guess where the cell will divide. However, in barnacle embryos the first division is not symmetrical. The embryo divides into two cells; the first is small and contains the granular yolk and the second is large and contains a little of the granular yolk but also contains all the globular yolk. In the picture I have pointed out the two types, but you can also see the different sizes of the cells. So even though we typically know where the spindle poles are, we don’t know if the top quarter of the cell will become the smaller cell or the bottom quarter. So far it has been frustratingly hard to find a cell the divides where we can see the cleavage furrow. So, part of what I am doing now is looking at videos that have been taken and taking pictures of embryos in different positions so hopefully we can find a pattern that will let us choose the best embryos to view. While the video can only capture what is happening on and right below the surface of the side of the embryo closest the camera, the pictures let us see about half way through the embryo. The picture below is one of the ones I took on Thursday using the utrophin probe.
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Hi everyone! This week I finished my aPKC GFP probe and tested it for the first time on Thursday! It has been incredible to work through all the steps necessary to create a probe and then to end up with something that could be tested. One part of being a scientist that I am discovering is that sometimes what you are testing works perfectly with your hypothesis; but other times you can end up with a result that doesn't. While the probe did express on Thursday it didn’t begin to show up visibly until the eight-cell stage and even then, the probe was hazy. We were hoping to see it clearly between cells. The two places that the probe did show up clearly were in the nucleus and in small circles that we think could be vesicles. Unfortunately, neither of those were places where we wanted to see it! So, the next step we tried was to increase the amount of RNA that we inject into the embryos. On Friday we injected three times as much RNA as we did the day before. The embryos seemed to handle the higher dosage just fine, but the same dimness and lack of defined location in the cell was still observed and the probe was still not well defined by the time that cell polarization or first cleavage happened. However, when we looked at the cells that we injected on Thursday the probe was working. We could see it in the cell junctions. So, the probe is working, it just isn’t working at the stage in cell development that I need it to. There is a possibility that the probe only shows up briefly during first cleavage, so we just aren’t catching it when it is lighting up. I will be continuing to take videos of the injected embryos to see if we can see anything happening.
This week I thought that I would show you what I am looking for when I am watching the barnacles. Image 1 is a barnacle searching for a mate. The long thin projection is the penis that he will probe the other barnacles with as he tries to find a barnacle close to him to fertilize. Image 2 is a successful fertilization. You can actually see the thin thread of sperm as it moves from the donating barnacle to the receiving barnacle. Image 3 shows another way to tell if a barnacle has been fertilized. It will release a cloud of excess sperm 10-20 minutes after fertilization. The pink is from the colored pencil that we use to keep track of the barnacles. After I see a mating or a release of sperm I wait until the animal’s feeding appendage is out again before retrieving the brood. Then I pull apart the brood so I can inject the embryos. Hi everyone, we are now on week 4! This week I have seen some awesome things in the lab. On Thursday I injected a new probe into barnacle embryos. It wasn’t the probe I have been working on creating, but it was one that Dr. von Dassow wanted to test. This probe attaches to histones, which are what the DNA is wound around in the chromosomes. We wanted to see if we could get the probes to change from emitting green light to red light when we shone a violet laser on them. It took awhile of playing around with the laser to get the protein to change color but in the end, it worked! This could let us track if a certain cell always makes the same part of an barnacle. An update on my research project is that the probe is getting close to being finished. I am hoping to start injecting it next week. I have also been working on getting videos of the embryos contracting. These videos are not taken under the confocal microscope but are allowing me to observe what is happening to the embryos when they move. I have been trying to get videos of the embryos when they first start making the wave contractions but so far, they have proved to be to delicate. I am happy to report that I have been practicing getting the embryos lined up on the injection dish and have gotten much better at it! This past weekend the OIMB REUs and the main campus REUs all camped together. On Saturday just the OIMB REUs spent the night. Although we were planning to just go tide pooling on Sunday morning, the tides were so low that we ended up going out on Saturday morning too! On Saturday we went to North Cove and saw many different types of algae, lots of worms, and some brittle stars. On Sunday afternoon we met up with the main campus interns and we enjoyed playing Frisbee and talking around the campfire. When we went tide pooling the next morning, we went to South Cove. There was less algae here and more sea anemones and hermit crabs. Dr. Watts even found an octopus! On Tuesday half of the interns gave a presentation on their research proposal. These presentations were more filled out and detailed than the previous presentations that we have given. Four weeks of chewing over what I am doing and will be doing and what I want to accomplish has made it where I can explain my project in a much clearer, concise way. I know that our presentations were all much clearer and easier to understand this time. Welcome to the week three blog post everyone! For me this week has still been full of learning new and interesting things. I have continued learning about and working with PCR. Polymerase chain reaction is used to make many copies of certain strands of DNA so there is enough DNA to work with. I’ve also been learning how to use the confocal microscope. I was able to get some beautiful and interesting videos of first cleavage with Dr. von Dassow directing me. I also think I’ve got the timing down with the barnacles. Up until this point I usually have had to wait until right before lunch to get a brood so the embryos don’t always have enough time to mature before the end of the day. I also found something that I need to work on. On Friday I tried pipetting some embryos into an injection dish. After watching Dr. von Dassow easily perform this maneuver perfectly I figured that I could at least get the embryos lined up on the dish. Well, I vaporized the first 20 I tried to move and got the next 20 stuck in the pipette. After that I got some embryos that were in one piece on the injection dish. However, they were scattered randomly across the dish. I’m going to make sure to practice before I try that again. We’ve also been busy with events outside of our lab. On Saturday we spent the day on and around the University of Oregon’s main campus. We were able to tour a lab that works with brain activity during movement. Also, since Thursday was the 4th of July, most people had little or no work to do in the lab. In the morning I was able to check crab traps with Renee and Megan. It was very muddy but I enjoyed learning about crabs and their habitats. In the afternoon we had a potluck at Sunset bay. It was a beautiful warm day, perfect for being on the beach. I enjoyed getting to climb down to visit a hidden beach and exploring the many hiking trails that weave through that area. We have our proposal for our projects due on Tuesday so I’m spending plenty of time looking up articles. Putting together a paper has given me the chance to more firmly decide how I will answer my research questions and what I need to do in order to get the final result. We are going camping this weekend with the interns from the main U.O. campus. I am looking forward to getting to know them better and hopefully enjoy a few hikes. Hi everyone! This is week two and I’ve got a project! On Monday Dr. von Dassow, my lab mates, and I discussed what we would be doing for our projects. I will be working with barnacles; Balanus glandula is their scientific name but informally they are called acorn barnacles. They are the most numerous barnacles on the Oregon coast and usually grow on docks and rocks. I will be studying cell polarization in barnacle embryos. In C. elegans cell polarization happens early, during first cleavage. When an egg is fertilized it starts dividing to create new cells. When the egg makes its first division it pushes some types of proteins into one half of the cell and other proteins into the other half of the cell. So, when the cell divides into two cells, one cell has a lot of some proteins and the other cell has a lot of other proteins. This happens because different parts of the creature will be formed from each cell. For example, part of one of the cells will eventually form the gastric tract. This cell will take the proteins that it will need to make this system. The moving around of substances is called cell polarization. I will be trying to discover if cell polarization happens in basically the same way in Balanus glandula as is does in C. elegans. To do that I will be using a probe to track atypical Protein Kinase C. The aPKC is found in many different organisms so there is a good probability that it will be found in Balanus glandula. I will be using a probe to track this protein and see if it will be doing anything interesting in barnacle eggs. Right now, I am practicing injecting substances into barnacle embryos and learning how to use the confocal microscope. This week I attached a video so I could show you what the barnacle embryos look like after a successful injection of RNA. What you will see is a bunch of white lines “flowing” through the embryos. The probe that was injected is a protein that sticks to the front of forming microtubules. When the microtubules stop growing, they either attach to a solid object or catastrophe which means it breaks apart. The video is sped up and is made up of pictures taken at different depths in the embryo. Helping to take this video was my introduction to working with the confocal microscope. If you watch the video closely you can see the cells dividing. On Sunday a group of the Interns went to Bastendorff beach. It was warm enough that we were wearing shorts and t-shirts! I know that I also enjoyed our meeting on Wednesday when we all gave a 5-minute talk on what our projects would be on. It was interesting to learn about everyone’s plans in more detail than just snippets from mealtime discussion.
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AuthorHi! My name is Sadie and I just graduated from Central Oregon Community College in Bend Oregon. I am working in Dr. von Dassow’s lab and I am excited to learn about research and cells. Archives
August 2019
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