An inducible and reversible genetics platform for novel drug target discovery

• Main Author: Leong, Mei Ling
• Other Authors: Klaus Erik Karjalainen
• Format: Theses and Dissertations
• Language: English
• Published: 2019
• Subjects: DRNTU::Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/87335; http://hdl.handle.net/10220/48048
• Institution: Nanyang Technological University

Understanding gene function provides insights into biological processes and disease pathologies. Such knowledge can be applied to benefit human health and medicine. With this in mind, I have established a genetic platform in mouse to study gene function. The platform consists of two transgenic mouse strains that were generated inhouse, through the use of CRISPR/Cas9 gene editing technology: (1) a strain that ubiquitously expresses the reverse tetracycline trans-silencer (rTetR-KRAB) in all tissues and (2) a strain where the tetracycline response element (TRE) has been inserted in close proximity to the target gene’s promoter. With appropriate double transgenic mouse strains, rTetR-KRAB binds to TRE in the presence of doxycycline and represses the target gene. Repression is dose-dependent and reversible. To demonstrate the feasibility of the platform, I have chosen to target Tcf7l2 which is a transcription factor that acts downstream of Wnt signalling. The Wnt signalling pathway has been implicated in colorectal cancer, but there are contradictory views on whether Tcf7l2 acts as an oncogene or tumour suppressor gene in this context. Preliminary data obtained using small intestine organoids derived from double transgenic mouse, show the importance of Tcf7l2 for intestinal homeostasis and ApcMin-associated adenoma maintenance. To expound this, I will utilise the Tcf7l2 transgenic strain to investigate if physiological rebalance of this gene has therapeutic effects in colon cancer. This platform can also be generally useful to study the physiological or pathological role of other target genes.