School of Science Division of Life Science 26 In Depth Characterization of Novel Cell Cycle Regulators in Cancer Cells Supervisor: POON, Randy Yat Choi / LIFS Student: THEN, Claudia Regina / BCB-IRE Course: UROP2100, Spring In this study, we investigated the effectiveness of combining the tetracycline-controlled transcription system with the auxin-inducible degron (AID) system to conditionally deplete gene in Chinese Hamster Ovary (CHO) cells. Specifically, we used the Tet-off system, which is regulated by the presence of Dox, and the AID system using AFB2 gene, which is regulated by the presence of IAA. To assess the conditional depletion systems, we targeted the AID-GFP gene in the CHO-tTA-AFB2 cell line after treatment with Dox and/or IAA. The results revealed that the addition of Dox only partially depleted the AID-GFP gene, while the addition of IAA did not induce AID-GFP depletion. In addition, a mAID-H6 standard curve for measuring mAID-GOI concentration in Western Blot was made. CRISPR/Cas9 Analysis of Essential Genes Supervisor: POON, Randy Yat Choi / LIFS Student: MEHTA, Mridini Prashant / BIOT Course: UROP1000, Summer The efficacy of a cell line expressing a fusion nanobody under Tet-On control plus an anti-FLAG frankenbody was explored. Doxycycline can be added to induce the expression of the minimized auxin-inducible degron fused nanobody which can then start frankenbody degradation when synthetic auxin, indole-3-acetic acid, is added. It was found through antibody assays that the addition of drugs can successfully induce frankenbody degradation in comparison to cells grown without drugs. Co-immunoprecipitation where HA was pulled down shows correct interactions in regards to green fluorescent protein (GFP) which proves that the complex is working in that particular aspect. Problems found include the inability to blot myc-AFB2 and leaky degradation caused by the original mAID-vhhGFP. Future steps include repeating immunoprecipitation through the separate pull-downs of HA and GFP with larger cell lysate quantities, reintegrating modified components into the cell line for reduced leaky degradation and most importantly, the introduction of FLAGtagged proteins. CRISPR/Cas9 Analysis of Essential Genes Supervisor: POON, Randy Yat Choi / LIFS Student: PARK, Junhyeok / BIEN Course: UROP1100, Spring Cyclin Bs are pivotal regulators of mitosis. The specific functions of the two major cyclin Bs, cyclin B1 and cyclin B2, remain incomplete understood. In this study, I investigated the relative expression of cyclin B1 and cyclin B2 in normal and cancer cell lines. HeLa, RPE1, and NIH/3T3 were treated with nocodazole to block cells in mitosis, before the relative expressions of Cyclin B1, Cyclin B2 were investigated using Western Blotting. The results show that HeLa has the highest B1/B2 ratio, followed by RPE1 and NIH/3T3. Therefore, the result suggests that cancer cells may have dysregulated Cyclin B1 in mitosis, and mouse cells have lower Cyclin B1 and B2 expression levels compared to human cells.
RkJQdWJsaXNoZXIy NDk5Njg=