Week 8: University of Iowa Neuroscience Program

August 6th, 2012

Kyle Durgin, ’14, Dimensions Fellow in Research

Wow! I find it hard to believe that I just completed the final week of my fellowship. It is hard to summarize all that I learned and experienced and the gratitude I feel toward all the members of the Cornell lab at the University of Iowa and to the Dimensions program at Cornell College. It was wonderful to be accepted as part of the research team in the lab and I truly appreciated the time and effort that everyone at the lab invested in answering my questions, familiarizing me with the lab, and working with me to give me an educational, rewarding summer. Throughout the course of my fellowship many of the questions I had about the future possibilities for my education were discussed and some of them were even answered. I also discovered that I really like the University of Iowa and I have the opportunity to continue my education there after completing my studies at Cornell. Prior to this summer I was determined to go to medical or graduate school somewhere outside of Iowa, but I can officially say those plans have changed thanks to my fellowship!

Working in a well-equipped, advanced lab gave me a much better understanding of scientific research. It was nice to be able to repeat things that I learned, like running agarose gels, isolating DNA, and other common laboratory practices in the lab courses at Cornell with Craig Tepper. This time however, I have done these things enough to have successful results. Working with living organisms was also a great experience, because it made the connection between scientific research and healthcare much clearer. It was fascinating to be able to expose the zebrafish to different treatments and then to be able to visualize the effects. Because a great deal of the research I did was conducted on zebrafish during their embryonic stage, I was very glad to have taken the developmental biology course at Cornell. Also, what I had learned in genetics was invaluable.

This last week I spent working on an in situ hybridization using an RNA probe to label the TRPM1 mRNA in zebrafish at 3 and 5dpf. Unfortunately, this was not successful, because I realized that I was using a “sense” probe which is identical to the mRNA sequence and not the “anti-sense” probe which is complementary to the mRNA. This was a good discovery because the probe had been used previously and had been deemed “no good.” I started the process of converting the sense probe into anti-sense for use in the future.

Although, officially this was my final week at the Cornell lab, I am looking forward to going back to finish off a couple of my experiments and touch base with Robert Cornell who has been enjoying a well-earned vacation in Colorado for the last week. I also plan on staying in contact with Rob and the others in the lab because I am sure that my relationship with the lab team will be useful in the future if I ever need advice or help with anything.

The end of this fellowship is bittersweet. I will miss everyone in the lab because we really had a fun time together. However, I am also very thankful that I was given this opportunity and I am also eager for my last full year at Cornell.

Week 7: University of Iowa Neuroscience Program

July 27th, 2012

Kyle Durgin ’14, Dimensions Fellow in Research

This week proved to be another fun week in the lab. I started a couple new experiments and other things this week which was enjoyable. I carried out a study on a batch of the zebrafish that lack trpm7 channels. These fish are peculiar in that at 3 days post-fertilization they experience paralysis that usually lasts just about 24 hours. Other studies have shown that by exposing the circulatory system of these fish to aqueous solutions containing different divalent cations can reverse this immobility in less than an hour. I was curious to see what effect, if any, calcium would have on the recovery of these fish. In order to test this I decided to expose the circulatory system of the fish to solutions containing a normal amount of calcium, an elevated amount, and a solution completely lacking calcium. I determined that the easiest way to open up the circulatory system was to amputate the tails of the fish using a scalpel. I then was able to test the response of the wild type fish compared to the mutants and the amputated compared to the unamputated. I observed a slight recovery in the fish exposed to higher calcium concentration. However, I think to see a more noticeable recovery, I will need to expose the circulatory system more and possibly increase the amount of calcium in solution. I continued the experiment overnight by putting the fish in individual wells under the time-lapse camera. The time-lapse device generates periods of light and dark and the change to the light period startles the fish and the camera records the movement of the fish. What I learned from this was that the unamputated wild type fish in the calcium-free environment moved much less than the ones in the higher calcium solution. It was cool to see such a clear difference in response based on the surrounding environment of the fish.

I also spent some time preparing for my work next week where I will be attempting to clone the transcript for trpm1 into a vector so we will be able to over-express the trpm1 protein in embryos. Due to the large size of the gene (5.6 kb) I have to use several sets of primers to amplify different segments of the gene. I will then marry the sections before I can clone it into the vector. I am quite excited to work on this!

Sadly, I had to say goodbye to one of the lab members this week because she was leaving to go home for a few weeks before classes start back up at the university. We had a wonderful potluck goodbye lunch/party for her. I brought enchiladas and brownies and there was a large variety of other delicious food items. My favorite thing was a watermelon that had been cut to look like a monster or some creature with teeth and then filled with other fruit. I got Christine to take a picture with it.

I was also hoping to ride my bike on Friday with RAGBRAI, but that didn’t work out because I was busy in the lab and at my other job. It was fun to see all the cyclists biking through Mount Vernon before I headed to the lab on Friday though. I am planning on participating in a large stretch of RAGBRAI next year!

Week 6: University of Iowa Neuroscience Program

July 20th, 2012

Kyle Durgin ’14, Dimensions Fellow in Research

I find it hard to believe that the 6th week of my fellowship is already over! Fortunately, it was another productive week. I took multiple photographs of the fish I had injected with the trpm1 morpholino and then treated to remove embryonic melanophores. I was then able to analyze the results by counting the stem cell melanophores in the wild type and morpholino-injected embryos. I determined that there was not a significant difference between the two groups, which, although it was a negative results, was good because it showed that the loss of trpm1 does not prevent the regeneration of melanophores from stem cells. I was also able to confirm the effectiveness of the morpholino by amplifying trpm1 cDNA from both wild type and morpholino-injected embryos using primers that straddle the morpholino site on the trpm1 transcript. When I ran the PCR products out on a gel, I observed an absence of the trpm1 band. I took this to mean that the morpholino is successfully knocking down the trpm1 gene.

I also started an in situ hybridization using a trpm1 probe to look for the location and quantity of trpm1 transcripts in 3dpf and 5dpf wild type embryos. I am excited to look at the embryos next week under the microscope to see if the hybridization worked. The probe that I am using hasn’t been successful in the past, but I am hopeful that it will work!

On Wednesday evening this week I also had the privilege of going to the Great Jones County Fair in Monticello to watch the truck and tractor pulls. It was a new and unusual experience for me. I also ate my first corn dog and I must admit it was delicious! I never experienced a fair growing up in the Catskill mountains of New York and I have really enjoyed going to the Iowa fairs the last few years. It is quite the sensory experience with the smells (fried food, tractor exhaust, sweat, and beer), sounds (music, carnival rides, tractors, and talking), and sights (lights on the rides, all the booths, and an incredible variety of people). I was also really impressed by the one-man band who covered several classics.

Week 5: University of Iowa Neuroscience Program

July 16th, 2012

Kyle Durgin, ’14, Dimensions Fellow in Research

I had a very successful fifth week here in the Cornell lab at the University of Iowa. The senior graduate student working in the lab here was kind enough to inject a bunch of zebrafish with the trpm1 morpholino for me. This was extremely helpful because she is very efficient at injecting the embryos and as a result I ended up with many more surviving embryos than when I had injected on my own. This enabled me to complete the first part of my experiment to determine the necessity of trpm1 and trpm7 in the regeneration of stem cell melanophores after the death of embryonic melanophores in zebrafish. I will analyze my data next week and hopefully I will be able to use my results to design the next step in my experiment.

The weather has been ridiculously hot and humid this past week so I was lazy and opted not to bike home at all. One of the graduate students on rotation in the lab is planning on biking with RAGBRAI when they do the stretch starting in Cedar Rapids. I may join her for that which would be very fun! I will have to see closer to the date though to see if it will work with my schedule.

It has been really cool to have Barbara Christie-Pope helping out with the research here in our lab this past week too. It has been fun to work with Barbara in the lab setting. Up to now I have only interacted with her in a classroom setting where she is feared by her students because of her challenging exams and assignments.

Week 4: University of Iowa Neuroscience Program

July 8th, 2012

Kyle Durgin, ’14, Dimensions Fellow in Research

Unfortunately, I wasn’t able to spend very much time in the lab this week due to the Fourth of July falling on a Wednesday. One of the difficulties about doing research with zebrafish or any living organism is that an experiment, especially one that studies some aspect of development, requires daily observation. This was part of my problem last week, because the developing zebrafish embryos require daily “clean-up.” This involves removing any debris (dead embryos, broken yolks, and chorions), replacing the water in the petri dish, and studying the embryos to ensure that development is occurring normally. Occasionally morpholino treatment is toxic to the embryo which is something that one would want to observe in order to determine what the correct dose should be.

I feel very comfortable working in the lab now and I really enjoy interacting with my colleagues. I really enjoy talking to the graduate and undergraduate students about what their plans are for their careers or further schooling. It makes me eager to plan how I want to continue my education after Cornell. A lot of people in my lab have been very interested in the BMB 485 class Craig Tepper teaches in the Bahamas (now Belize). All of them have said: “Wow, I wish my school would have offered an opportunity like that!” Hearing that from people from other schools makes me really happy to have chosen Cornell and I must admit, I am very excited to go to Belize next year.

This week I repeated an experiment I had attempted earlier this summer without much luck. We have a line of zebrafish, nutria mutants, that have a heat-sensitive trpm7 gene. When these fish are exposed to warmer conditions (34 degrees C), the gene is not expressed. At a lower temperature (28.5 degrees) the gene is expressed. I was hoping to see at what time point the heat treatment would cause the fish not to develop melanophores. I was unsuccessful however, because some of the fish didn’t breed and the ones that did, all died by 48 hours. This may have been a result of me not being able to clean out their dishes on July 4th. I will have to repeat the experiment and hopefully the fish will behave!

Week 3: University of Iowa Neuroscience Program

July 2nd, 2012

Kyle Durgin, ’14, Dimensions Fellow in Research

I can’t believe that June is already over. This summer is flying by and now that I have completed the third week of my fellowship here at the University of Iowa, I am realizing that research isn’t something that can be rushed. I have been refining my experiment for the fourth or fifth time now in hopes of answering the question I was presented with during my first week here: “Is TRPM1 required for the regeneration of melanophores from stem cells after the death of embryonic melanophores?” In theory, this question should be easy to answer. By knocking down the TRPM1 expression in zebrafish embryos, then treating the embryos with a chemical to kill all the embryonic melanophores, and finally washing the chemical off the fish and allowing them to develop, we should be able to see if TRPM1 plays a role in the regeneration of the melanophores. The problem is, with two consecutive treatments, initially the TRPM1 morpholino and secondly the chemical, it is asking quite a bit of the fish to survive and continue development. I have been continually adjusting my method in order to figure out the most effective treatments to use to have the best results. I believe that I have been learning a great deal about the scientific method and have been really enjoying the work here.

It was nice to have everyone back in the lab after the zebrafish conference. There has been a lot of excitement from the people that went about the new methods and research tools that were presented at the conference. In our lab meeting on Friday, the members of the lab that had attended the conference presented the things that they had found most interesting. It was cool to hear about the different advances that have been made in zebrafish research and I am hoping that some of what they learned about tools to study zebrafish will be seen in the experiments that are being done in our lab. It was also great to see Barbara Christie-Pope on Friday. She visited to catch up with everyone at that lab because she worked here last summer. I was able to update her with what I have been doing. I was also happy to hear that she is hopefully going to be doing more research here this summer starting sometime in mid-July.

The weather has been insanely hot and it is so humid too! For that reason, I have been hesitant to ride my bike back from the lab. I am hoping it will cool down this upcoming week because the ride home is quite lovely in the evening. I also was able to float down the Maquoketa River in by Monticello this weekend which I highly recommend. I went with some fellow Cornellians and some other people that are doing research in the labs at the University of Iowa this summer. It is an experience that I highly recommend especially on these hot summer days! I am eagerly looking forward to see what the month of July might bring!

Week 2: University of Iowa Neuroscience Program

June 23rd, 2012

Kyle Durgin, ’14, Dimensions Fellow in Research

The second week of my fellowship went by even faster than the first week despite the fact that literally everyone except one of the graduate students on rotation was at an international zebrafish conference for the second half of the week. Fortunately, everyone had done a good job introducing me to the lab because I was able to get a decent amount done on my own.

I have been working on repeating my initial experiment with knocking down the trpm1 genes in the wild type fish using a trpm1 morpholino. I slightly changed my injection techniques and have been having much more success injecting the correct amount of morpholino in the correct location in the zebrafish embryos. I also used some minipreps to isolate DNA and RNA and as I worked through the miniprep protocols I remembered the labs with Craig Tepper where we used the same kits. Having previously done these kits at Cornell, I had no problem doing them alone this last week. Also, the pipetting skills that I developed in the lab courses with Craig Tepper, Barbra Christie-Pope, and Jeff Cardon are serving me well!

In order to make the commute from Iowa City to Mount Vernon, I decided to bring my bicycle with me and ride the 24 miles back to my apartment. One of the big advantages to car-pooling (besides not paying for parking) is that I can skip the drive home and ride my bike. This saves me from being sweaty when I get to the lab and is good exercise. And to all the haters that say Highway 1 is dangerous, I have not had any problems….yet.

Every day I spend working at the University of Iowa makes me more and more convinced that it is where I want to go to school after I am done at Cornell. I have spoken to a few people about the MD/PhD program at Iowa and it sounds very enticing although it would be a very challenging program to get in to. I am looking forward to everyone returning from the zebrafish conference next week and I am excited to continue doing research.

Week 1: University of Iowa Neuroscience Program–Iowa City, Iowa

June 21st, 2012

Kyle Durgin, ’14, Dimensions Fellow in Research

My first week here at the University of Iowa has been fantastic. The parking in Iowa city is still a challenge and the bus routes still confuse me, but I have been car-pooling with some other Cornell students which has made my life a lot easier. The members of the lab team have been amazingly helpful with orientation around the lab and helping me get involved in the research. I was fortunate to have done some reading in regards to the research being done in the Robert A. Cornell Lab prior to the start of my fellowship which has helped me dive right in!

The main research in the lab has been done on zebrafish which are a great model for studying vertebrate development and genetics because of the ease with which they can be studied. It is fascinating to be able to watch the eggs develop from the single cell stage to a fully formed fish in a matter of days. The observable cell division and differentiation due to the transparency of the fish enables us to target specific genes within the fish and study the effects of knocking down or knocking up the gene.

So far my goal has been to focus on a certain family ion channels known as TRPM (transient receptor potential melastatin) channels. These channels function in a wide variety of physiological functions and dysfunction of different members of this family has been related to numerous diseases. We hope to gain a greater understanding of how these channels function in zebrafish in hopes to eventually relate our findings to diseases in humans.

I spent my first week was spent learning different essential lab techniques in regards to working with zebrafish. I first learned how to differentiate between the male and female zebrafish, as this is important to get the fish to breed. I then learned how to inject the embryos at the 1-4 cell stage. The injection process requires steady hands and a fair amount of patience and although my first couple hundred injections were actually surprisingly successful, I know that I could use a lot more practice! Once I had mastered the injecting, I went on to start my first experiment to determine if the first TRPM channel, TRPM1 is necessary for the re-development of melanophores (pigment cells) from stem cells following the death of the embryonic melanophores.

So far, I have been very grateful for the previous lab experience I received in the numerous science courses that I have taken at Cornell, specifically cell & molecular biology, genetics and biochemistry. The lab techniques covered in those courses have been invaluable for helping me start my summer here in the Anatomy and Cellular Biology Lab. I also was very fortunate to be able to talk to the graduate students working in the lab about graduate and medical school which has already helped me start to shape my plans for when I am done at Cornell. I am eagerly looking forward to next week and what it might bring!

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