School of Science Division of Life Science 13 Molecular Regulation of Skeletal Muscle Stem Cell Quiescence and Activation Supervisor: CHEUNG, Tom / LIFS Student: KARAMCHANDANI, Gia / BIBU Course: UROP1100, Spring Genotyping works by analyzing DNA to determine genetic makeup, allowing for the investigation of the molecular mechanisms that regulate biological processes. Genotyping of the Pax7creERT2 and ROSA26MetRSGFP mouse lines was conducted to confirm the presence of particular DNA sequences. PCR amplification and gel electrophoresis were utilized to detect specific DNA sequences corresponding to each gene's wildtype or mutant alleles. The results provide a base for further extension into the function and role of these genes in muscle stem cells. Additionally, laboratory techniques and technologies, such as cell culture, fluorescence microscopy analysis, and cryopreservation, can be used to study the different aspects of muscle stem cell behavior and function, enhancing the understanding of muscle stem cell biology. Molecular Regulation of Skeletal Muscle Stem Cell Quiescence and Activation Supervisor: CHEUNG, Tom / LIFS Student: NGAI, Man Yee / BCB Course: UROP1100, Spring This study aimed to optimize the protocol for investigating the role of miR-653 in regulating the quiescent state of stem cells through fluorescence-activated cell sorting (FACS). Adult muscle stem cells were used as the model system, and the protocol was optimized based on previous research. This report presents the progress achieved in optimizing the protocol, although biological replicates were not included in the experiments. We compared the concentrations of antibodies targeting Pax7, Ki67, GFP, and MyoD after 36 and 48 hours. AntiPax7 and anti-Ki67 antibodies did not yield observable results. Anti-GFP exhibited optimal performance at a concentration of 1:100, while anti-MyoD did not show any significant differences among the tested concentrations. Furthermore, no significant reduction in stem cell activation was observed when different concentrations of EdU and Azide were tested. Additionally, horse serum prepared at lower temperatures tended to result in reduced stem cell activation. Molecular Regulation of Skeletal Muscle Stem Cell Quiescence and Activation Supervisor: CHEUNG, Tom / LIFS Student: PARK, Changmo / BIOT Course: UROP1100, Fall Observation of whether stem cells are affected by a specific gene or a microRNA pathway can be done with the help of the RNA interference technique. After the knockdown, phenotypical changes such as the number of stem cells and other expressions of proteins can be easily detected and analyzed with the help of immunofluorescence. Satellite cells are one of the stem cells which play an essential role in maintaining muscle fibers and repairing skeletal muscles by proliferation, self-renewal, and differentiation. In this paper, molecular and genetical methods of the siRNA/miRNA transfection to the satellite cells and the experimented and utilized genes such as the YFP, CKS1B, and MyoD are discussed.
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