Alina Schmidt and Dr. Laura C Bridgewater
Bmp2 is an important growth factor that regulates many stages of development including the development of the heart, muscle, and bone. A novel nuclear variant of BMP2 was discovered before my addition to the lab and studies began to determine nBMP2’s function. Of main importance to my project was the creation of nBMP2 chimeric mice.
Chimeric mice contain either the normal or mutant version of BMP2 in their individual cells. This means that all the mice contain the mutant version of BMP2, but not all of the mice may have mutant BMP2 in their germ line. The chimeric male mice were bred with wildtype (normal) black 6 female mice to produce prodigy heterozygous for mutant BMP2. Out of the 9 Original chimeric male mice only 7 of them produced prodigy with the mutant construct. Once the first generation of heterozygous mice were old enough, crosses were set up to produce litters containing pups of all three genotypes: homozygous wildtype, heterozygous, and homozygous mutant. I expected that the homozygous mutant mice would die during embryonic development, as previous studies on zebra fish showed mutant BMP2 was embryonic fatal.
The first litter was born Nov 3 2007. I observed the birth to make sure all the pups were born alive and that none of them died shortly after birth. All pups born were healthy. DNA samples were taken from each mouse and digested. The samples were then analyzed through PCR to determine the individual mouse’s genotype. Shockingly the results showed all three genotypes from the litter. That meant that the homozygous mutant BMP2 mice were not embryonic fatal but viable! Since the mutant mice were alive I now had to change the experiments on how to determine the phenotype. I then began many observations on the mice which looks for normal development from their adolescence through adulthood. No obvious discoveries were seen during their adolescence and early adulthood. I then crossed mutant females with mutant males to see if the mice were fertile and they were. Because of these results I kept the colony growing by crossing heterozygotes with heterozygote to make sure all three genotypes were produced.
With no obvious developmental differences I started experiments to determine differences in balance, gait, coloring, weight, strength and senses. With the demands of the growing colony and time needed to test and take care of the mice three other people were put on the project with me. The balance, gait, weight and coloring tests gave no significant results. Only minimal tests were conducted on the mice’s senses and should be further looked into. Fortunately the strength test did show a potential phenotype.
Once the mice were about a month old we started conducting a limb flex strength test on the mice. This consisted of the mice holding on to a wire mesh and timed for how long they held on. It was quickly obvious that there was a strength deficiency in the mutant mice. This experiment was performed on the mice for about 6 months consisting of over a hundred mice being tested. Analysis showed that there was a significant time difference between mutant male and wildtype male mice (Figure 1). There was also a difference between mutant female and wildtype female mice but it was not as significant as the difference between the males.
This led us running and swimming experiments on the mice. The swimming test gave showed the same results as the strength test but in a less quantifiable manner so we did not pursue that test. We then put a mutant and wildtype mouse from both males and females into individual cages with running wheels. There did seem to a few minor differences between the wildtype and mutant mice. The mutant mice seemed to take a few more breaks during their running and would also sometimes run during the day. However, no significant differences were shown.
Multiple dissections were performed on mutant and wildtype male and female mice. All major organs were compared but no significant results were found. Muscle samples were taken from running and non-running mice of all genotypes. Western blots were performed on these muscle samples looking to verify that no nBMP2 was found in the nucleus. Other western blots looked for any major differences in proteins dealing with muscle contractions and strength.
From synthesizing all the results it seems to be that the biggest difference in the mice is their lack of muscle strength. Through internet searches and reputable journals I found a study that shows muscle fatigue is caused by Ca+ leakage in muscle cells that are over worked. This happens because as the muscle cells are stressed some of the stabilizing proteins fall off and allow Ca+ to leak into the cytoplasm. nBMP2 may have an effect on these calcium channels during development. My future studies are focused on determining the cause of the muscle weakness.
The work from my project will be presented in the 2009 American Society for Biochemistry and Molecular Biology (ASBMB) conference under the Membrane Dynamics and Organelle Biogenesis division through a 15 minute oral presentation including a poster. Results will also be presented in the 2009 Utah Conference on Undergraduate Research at Westminster College.