Randall Roper, Microbiology
The research project I proposed entailed mapping the gene or genes that control susceptibility to Murine Experimental Orchitis by using a large backcross population of 110 animals. These animals have been generated by obtaining an Fl population from the susceptible SJL mice and resistant B lO.S mice and crossing the susceptible Fl population with disease resistant BlO.S mice. Through the use of a histological report of the susceptibility of the large backcross population to Experimental Allergic Orchitis, and the use of microsatellite and RAPD markers to identify distinguishing polymorphisms, the gene may be linked to a specific chromosome of the mouse genome and an estimate to the precise location on the chromosome may be given.
Isolation of the DNA
From tissue obtained from the mice of the backcross population, I isolated DNA from each of the mice. Two different processes were used in obtaining the DNA. The first process entailed a commonly used three day process of homogenization, extraction, and purification of the DNA. A newly developed process was used on approximately half of the tissues. This second process cut the time of the DNA extraction by a day and also gave a purer isolation of the DNA, free from protein contamination. The process of isolating the DNA from allllO mice and two parental strains has been completed.
Screening Microsatellite and RAPD Markers
The next step to achieve the goal of mapping the gene or genes responsible for the disease was to screen microsatellite and RAPD markers on each of the 20 murine chromosomes. First of all, markers for the chromosomes that have been previously linked to other murine diseases in our lab were selected because of their ability to distinguish polymorphisms in the parental strains of the mice. This involved carefully selecting markers so as to cover the entire length of the chromosome, testing through the use of PCR and gel electrophoresis, and distinguishing those markers which show a visible polymorphism on the parental strains. A recent publication about markers already found that distinguish polymorphisms in the B lO.S and SJL mice has helped my search greatly.
By repeating this rather complicated process many times for different microsatellite markers, I have learned how to succeed in performing this process and have become proficient in avoiding the mistakes which lead to no results obtained using this difficult procedure. I continue to map microsatellite markers to the remaining chromosomes of the mouse genome.
Mapping the Gene
After a histological report is made that distinguishes the susceptible mice in the backcross population, I will use the markers obtained to find the probable location of a gene or genes that may cause Murine Experimental Allergic Orchitis. This project will continue, as previously outlined until this final goal is reached.