This experiment will be done using C57BI/6 wild type mice from Professor Moore’s lab. To begin two dams will be mated per male sire over night for a total of 10 females with 5 males by 5 pm. We will then look for evidence of fertilization through identification of the post copulation vaginal plug by 10:00 am the next morning. If more than two copulation plugs are identified then we will increase the number of dams per treatment group starting by increasing the number receiving lithium carbonate injections, but still attempting to maintain the 1:1 ratio of saline injected to lithium carbonate injected mice. The animals will be housed in specific pathogen-free barrier housing in the basement of LSP. It is likely that two rounds of timed matings …show more content…
The dams are expected to weigh approximately 15-20 g so a total of 6 μg via 100 μl of 60 μg/ml lithium carbonate solution dissolved in phosphate buffered saline pH 7.2. Both treatment groups will receive intraperitoneal injections of 100 μl of solution, the control receiving the sterile saline and the treatment group receiving lithium carbonate, through a sterile syringe with a 25-gauge needle into the right lower abdominal quadrant. The injections will occur three times during pregnancy, embryonic days 2.5, 4.5 and 6.5. Professor Moore will then sacrifice the pregnant dams by cervical dislocation at embryonic day 9.5, the uterus will be removed from each and the embryos will be isolated. The number of embryos from each pregnancy can range from one to sixteen and is an uncontrollable variable (Theiler, 1989). After each embryo is isolated and separated from its placenta it will be weighed and imaged from analysis of crown-rump length and morphological …show more content…
The images will examined to determine if there was any disruption in the formation of the neural tube. The neural tube length will be measured and recorded. It will be measured using a camera attached to a microscope with a camera attached. The brain will be imaged with a ruler in view so the length can be determined. The magnifications used to get the best view of the neural tube will also be recorded so all the images can be compared. The brain tissue will then undergo a whole mount DeadEnd Fluorometric TUNEL assay following protocol from ProMega. This assay will identify the cells undergoing apoptosis by incorporating a fluorescent molecule at the 3’-OH DNA ends using the terminal deoxynucleotidyl transferase recombinant enzyme. The DeadEnd kit and materials provided will be used in addition to phosphate buffer saline, Hoechst, a DNA counterstain, and Dako mounting media. The TUNEL assay will require attaching cells to the microscope slide, fixing them using 4% methanol-free formaldehyde in PBS and washing them. We will them have to permeablize the brain tissue with Proteinase K, wash them, pre-equilibrate the cells and label the DNA strand breaks with fluorescein-12-dUTP and incubate for 1 hour. Then a stop reaction will be undergone, the cells will be washed again and stained. After the stain the cells will be washed again and then sample will be analysis. The signal will be visualized using the Leica SP8