Megan Bickner Jessie Savu Josh Fletcher Jon Nau 5 4 09 Background Migraine is a common neurological disorder that last on the order of hours to days and affects approximately 28 million people in the United States Advances in molecular biology have greatly clarified the pathophysiology of migraine though much research has yet to be done Of particular importance to this research is the role of Calcitonin Gene Related Peptide CGRP CGRP is neuropeptide that when injected into migraineurs often causes migraines and migraine like headaches to develop The presence of CGRP alone is not enough it must bind to the cell surface molecule Receptor Activity Modifying Protein 1 RAMP1 This triggers a signaling cascade in the cell which is thought to eventually up regulate CGRP expression creating a positive feedback loop that is consistent with the observation that the duration of migraines can be quite sustained In an effort to create a model system in the mouse a transgenic strain was generated through virus mediated gene delivery and subsequent breeding programs The gene introduced to the mice was the human RAMP1 This transgenic strain was engineered in such a way that the human RAMP1 gene is only expressed in the nervous system see fig 1 A variety of experimental methods have been developed to assay the behavior of mice when treated with noxious stimuli normally associated with pain such as heat excessive light or mechanical pressure Of interest to this study is the test of photophobia or light aversive behavior The experiment was performed by putting a mouse in a box that was well lit on one half and dark on the other half The wall separating the two halves had a small hole through which the mouse could travel The time spent in light was recorded in seconds and the person testing the mice was blind to their genetic background Fig 1 Introduction of the human RAMP1 gene into the mouse genome was engineered in such a way that a substantial quantity of protein is made and is present on the neural cell surface Questions 1 Do transgenic mice spend less time in the light than control mice 2 Does gender have an influence on time in light between transgenics and controls Hypothesis 1 Ho xc xt Ha xc xt 2 Ho xtm xtf xcm xcf Ha xtm xtf xcm xcf Statistical Tests We defined our groups of mice as two independant samples To determine whether transgenic mice spent significantly less time in the light compared to the control mice we used Student s t test We used ANOVA to test our second hypothesis to see if gender affects time spent in light We performed a further ANOVA with the Bonferroni correction Results Hypothesis 1 Time in light seconds Genotype Hypothesis 2 Time in light seconds Genotype Conclusion After conducting the statistical tests we found that we were able to answer our initial questions We wanted to know if insertion of the gene for human RAMP1 would cause the transgenic mice to spend less time in light when compared to the control mice that lacked the gene We also wanted to know what effect if any gender had on time spent in the light between controls and transgenics Using SAS we analyzed the data to test our hypotheses We used Student s t test for our first hypothesis and we found that transgenic mice do spend less time in the light t 3 97 P 0 0002 Next we used the ANOVA procedure to determine if gender influenced the time spent in light We found that there is a difference in the sample means F 6 57 P 0 0005 so we performed another ANOVA with a Bonferroni correction to see which groups may have been significantly different from each other We found that transgenic females spent significantly less time in the light than the control females however there was no significant difference between transgenic males versus control males The initial delivery of the gene into the ancestral transgenic mice was a rather difficult and at the molecular level a somewhat random process so we are unsure of the exact location of the transgene within the mouse genome Although unlikely it is possible that the gene inserted into another gene and that this disruption may account for the observed light aversive behavior
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