Kristopher Bennion and Dr. Laura Bridgewater, Zoology

In the beginning of the 1999 Fall semester I made a request for a scholarship for my efforts in research with DNA Recombination, specifically to locate enhancers along a defined length of the Collagen XI gene in Dr Bridgewater’s Lab. I desired a better understanding gene activation and regulation and what allowed only one specific gene to be activated of the thousands found within the cells DNA. In using the Collagen XI I gene, I was trying to use the information gained in hopes of coming closer to solving such issues as dwarfism and cleftpalate, which results from abnormal expression of this gene. You generously granted this request, for which I am very grateful. I would like to take this opportunity to report back to you and your department on what I have accomplished and learned through my research in this project.

The first major task for this project was to simply design my own primers. This was done to delete the selected sequence in the gene to prove which, if any, of the sequence acted as a enhancer in the expression of this gene. I simply took the known base sequence and removed certain segments of the gene. I then wrote this new sequence down, both the forward and reverse, and ordered them to be made for me. Through this process I developed the necessary primers to test the region of the collagen gene I was working on. When I received these primers I was ready to proceed on to my next step, PCR.

The purpose for PCR was to amplify, or in other words, create a large number of DNA copies of the part of the gene that I was not deleting. This was a very slow process at first for many reasons, the greatest of which was that it marked my first time doing this type of work and I didn’t want to make a mistake. I carefully followed the instructions for this procedure and was pleased to see that the DNA, PCR product, was correct. I then moved on to inserting this DNA into my plasmid. This is where I ran into problems, which drastically slowed down my project. The first step in this process was to remove base pairs from the ends of my DNA to create sticky ends, so that the DNA could be inserted into a pre-made plasmid. There was no way to test these results but to simply proceed with ligation and hope the digestion was done correctly.

Ligation was done to insert my DNA into the pre-made plasmid upstream of the B-galactosidase, so that I could test the effect of my deletions on gene expression. Transformation, the act of inserting the newly made plasmid into bacteria to grow a large amount of the plasmid, was then preformed. I was finally able to test my plasmids to see if they and the DNA had joined correctly. I was to grow up Bacteria with the plasmid and then plate them on an ampicillin (Amp.) positive agar plate. Only the plasmids that carried the Amp resistance gene and that had formed a complete circle by ligating to both ends of my DNA insert would grow on the plate. I would then remove our plasmid from the bacteria.

Unfortunately, when I plated my bacteria which were suppose to contain my plasmid, no colonies appeared. I repeated the process several times, trying to correct the problem with no success. Neither Dr. Bridgewater nor I could determine the problem. Either no Bacteria would grow or I would get colonies but when I went through the process of purifying out the plasmid nothing, would be present. The two results I was receiving contradicted each other and caused a lot of questions. I was eventually able to determine that I had been using a large DNA construct instead of the correct plasmid. I quickly rectified the problem, by starting over from PCR one more time and eventually was able to insert my primers into the correct plasmid for testing in transfection.

At this point I turned over my research and plasmids to a fellow student, Steve Smith, who continued the work and preformed the actual transfection. He found that two of my deletions reduced activity in the gene’s expression, showing that these parts of the gene have potential enhancer sites. My fifth deletion reduced activity, which was expected, because this deletion removed a previously known enhancer element. What we did find though was that my sixth deletion reduced expression as well, indicating that a new enhancer is found within this region. We will now have to locate exactly where this enhancer element is found within this deletion. We all also move on to test the new enhancer for independent chondrocyte-specific enhancer activity in tissue culture and eventually in transgenic mice experiments.

It is clear that there is a lot more to do with this project, and even more to be learned and uncovered. The process of experimentation in genetic recombination has been a very beneficial process for me. It has given me a better understanding of science and the true processes at work behind it. It is not simply to follow the recipe and wait and see the results, but a long and exacting process of questions, work, and experiments to be able to quantify and receive validation of your hypotheses. I learned patience and how to think about and solve problems, as well as answer question relating to my work. My skills in these areas have increased, as well as my knowledge in this growing and expanding field. I appreciate your support in my research and development of these skills.