Josh Yates and Dr. Laura Bridgewater, Microbiology & Molecular Biology
Introduction
Bone morphogenetic proteins (BMPs) contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors. Recently, researchers in the Bridgewater lab discovered a nuclear variant of Bone Morphogenic Protein 2 (nBMP2). To better understand the function of nBMP2, our lab generated a mutant mouse line in which nBMP2 is missing from the nucleus. These mice display learning and memory deficits, muscle dysfunction, and abnormal immune responses. In order to validate the mouse model and to study the molecular mechanisms of nBMP2 we have generated immortalized embryonic fibroblast cell line.
Methodology
Homozygous wild-type and homozygous mutant nBMP2 embryos were isolated from crosses of heterozygous mice at about 13.5 days of gestation. The embryos were dissected and disaggregated in cold trypsin overnight. The cells were resuspended and seeded in culture flasks while genotyping of each embryo took place. Cells were plated and passaged according to current protocols. Aliquots of each cell type were seeded in six well dishes and transfected with the plasmid pSV3-neo which constitutively expresses the SV40 large T antigen. Cells were passaged to 50 passages to ensure that immortalization had taken place. Cells were imaged using immunocytochemistry and confocal microscopy using a BMP2 antibody to stain for nBMP2 and topro-3 iodide was used to stain chromosomal DNA.
Results
Both cell types were imaged several times using confocal microscopy. Several of the wild type cells appeared to have nuclear staining with the BMP2 antibody, however because mutant cells looked similar to those of wild type mice, we were unable to draw conclusions based on these results.
Discussion
Recently in the lab two other students did similar work with muscle tissue from the mice and were able to definitively show a difference in staining between the wild type and mutant mice. This could be because the nuclear variant of BMP2 is not expressed in embryonic fibroblasts.
Conclusion
I would like to that everyone who helped out and supported this project. I would especially like to thank my Mentor Dr. Laura Bridgewater and those who make it possible for students to participate in creative research through the ORCA program at BYU. Although my project did not yield the desired results, I was able to learn much about cell culture, confocal microscopy, and molecular biology in general. The cell lines that were generated are still being used in the lab to carry out further experiments.