Download: Mémoire no 2497 Annexes Mme El Chammah.pdf (935.55 [Ko])
A Master's thesis.
Master (thesis) (master)
Talen technology used to produce a synovial sarcoma cell model
Université de Lausanne, Faculté de biologie et médecine
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Synovial sarcoma (SS) is to this day a devastating malignancy, of which we have very little mechanistic understanding. Two elements characterize SS: the cell of origin, a multipotent myoblast precursor cell, and its signature chromosomal translocation t(X:18) that generates the SYT-SSX fusion gene and corresponding fusion protein. The aim of this project was to produce a TALEN, a tool that could be used to produce SS cells de novo by introducing the translocation into a selected cell line (C3H cells). This may give us insight into the translocation's impact on the cell's physiology early in transformation. The TALEN, composed of a nuclease (Folk I) and an amino acid repeat module array, responsible for the specificity of the nuclease's cut point, was generated using a basic cloning technique. The targeted break point in the murine genome was determined following several criteria including its resemblance to human SS break point and the limitation of off-target effect. Damaged DNA produced by the transfection of C3H cells with the TALEN triggered homologous recombination (HR) thereby introducing the LoxP site into the targeted sequence. The lox P sites were then recognized and recombined by CRE thus producing the translocation between chromosome X and 18. Efficiency and specificity of TALEN cleavage must now be demonstrated by PCR screening. HR's specificity and efficiency, must also be established using T7 Endonuclease I mutation detection assay.
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