Dana L. Murphy and Dr. Laura C. Bridgewater, Zoology
Collagen fibrils play an important role in proper skeletal development. Defective collagen can cause human skeletal disorders such as osteoarthritis that affects an estimated 20.7 million Americans. A particular mouse line, housed here at BYU, contains a mutation in the Col11a1 gene. This mutation results in defective type XI collagen, which is an important component of articular cartilage, and eventually leads to osteoarthritis (1).
Dr. Laura Bridgewater and Dr. Robert E. Seegmiller received funding from the National Institute of Health (NIH) to study whether the phenotype caused by this mutation in the Col11a1 gene can be rescued through the expression of a non-mutated version of the gene. I proposed a research project in which I would focus on the first phase of their research grant, which was to create a Col11a1 transgene that is expressed only in mouse cartilage cells.
The first step in my project was to create the transgene vector. In order to construct the vector, I planned on obtaining the mouse cDNA Col11a1 from Dr. Hidekatsu Yoshioka, a researcher in Japan. However, due to unexpected complications in making the cDNA Col11a1, I was not able to obtain it from Dr. Yoshioka. Therefore, in order to continue with Dr. Bridgewater and Dr. Seegmiller’s transgenic mouse project, we turned to an alternative method of obtaining the gene. I am currently working on obtaining the Col11a1 gene from a Bacterial Artificial Chromosome (BAC) library. BAC libraries contain sections of DNA, including full-length genes. In order to determine which BAC(s) in the library contains the full-length Col11a1 gene, I am working on creating a probe that can be used to screen the library to find the gene. Once the correct BAC has been found, it will be used to construct the transgenic vector as originally planned.
In addition to this work, I have used the resources made available to me through the ORCA scholarship to focus on additional areas that supplement my original proposed research. I have developed a more reliable and straightforward method of determining which mice contain the Col11a1 mutation, referred to as genotyping mice in the colony. I have also studied mouse articular cartilage at the ultra-structural level with the transmission electron microscope.
Dr. Seegmiller has maintained the mouse colony for many years. Previously, through breeding and examining the off-spring, he has been able to determine the genotype of the mice. However, this method of determining genotypes is time-consuming and labor-intensive. Under Dr. Bridgewater’s guidance, I worked on a molecular method of genotyping the mice which is more reliable and objective. To do this, I obtained a one-centimeter tail sample from each mouse needing to be genotyped. The tissues were digested and the DNA was isolated. The region of Col11a1 was end labeled by radioactive, P32-tagged, primers, and the gene was amplified using the polymerase chain reaction (PCR). The PCR product was then electrophoresed on a polyacrylamide gel. This separated the DNA into bands in the gel. By exposing the gel to film, the radioactive Col11a1 gene created bands in the film that were then interpreted to determine the genotype of the mice. If a mouse were heterozygous, or contained one non-mutated gene and one mutated gene, two bands would appear on the film. If a mouse were homozygous, or contained two mutated genes, only the bottom band would appear. If a mouse were wild-type, or contained two non-mutated genes, only the top band would appear. This protocol, when carefully followed, gave reliable data needed to maintain the colony of mice. From my work on this protocol, I have established a better method of genotyping mice in Dr. Bridgewater’s lab.
I also studied mouse articular cartilage at the ultra-structural level. Our understanding of how mutations cause osteoarthritis is limited. Therefore, I hoped to gain insights as to how the defective Type XI collagen fibrils cause osteoarthritis by studying articular cartilage under the transmission electron microscope (TEM). Before the articular cartilage of the mutant mice could be studied, it was necessary to gain a better understanding of control, non-mutant, mice. I began my work with a control mouse. Using the TEM, I studied and obtained photos of several sections of articular cartilage. After obtaining this information, I began work on cartilage obtained from a mutant mouse. This work is ongoing. However, through my accomplishments in this area so far, I have discovered better methods of preparing tissue used in TEM work. These methods, I believe, will save time and energy for me as well as for those who follow me in this work.
Although I have had complications and have not yet been able to accomplish all of the original research I proposed, I believe I gained valuable experience and knowledge and have established protocols that will benefit my later work and that of my colleagues. I learned, when faced with problems, to evaluate what I did that could have caused the problems and to determine how to avoid the same mistakes in the next attempt. Overall, I have enjoyed my time in the laboratory. This research, provided through my ORCA scholarship, has been very influential in my future career plans. I have seen the potential benefits research such as this holds for the future and have decided to continue my involvement in it by pursuing a Masters Degree here at BYU.