Development of a new strategy for inducible and reversible gene knockout.

• Main Author: Tan, Hanrong.
• Other Authors: Klaus Erik Karjalainen
• Format: Final Year Project
• Language: English
• Published: 2013
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: http://hdl.handle.net/10356/53790
• Institution: Nanyang Technological University

The ability to inactivate gene function has contributed to functional genetics studies. Current techniques of gene knockout using Cre-loxP recombination and RNAi have achieved varying degrees of success despite certain limitations. To overcome these limitations, we initiated the development of a new strategy to achieve inducible and reversible gene knockout. The strategy requires two components, a tetracycline inducible repressor (TetR-kid) and a repressor binding sequence (ΔTRE). TetR-kid was obtained by coupling the Tetracycline repressor protein (TetR) to the Kid1 protein. ΔTRE is a modified tetracycline response element (TRE). Here, a Rosa26 targeting vector was constructed in efforts of developing this strategy. The vector was designed to introduce the TetR-kid transgene into the Rosa26 genetic locus to achieve ubiquitous and uniform expression of TetR-kid. The vector was obtained by modifying a BAC containing the locus. By recombineering, the TetR-kid transgene along with an ACN cassette for positive selection was introduced into the locus followed by a second recombineering step to retrieve the modified BAC fragment into a vector that contains a negative selection marker, a diphtheria toxin fragment a (DTA) gene. This targeting vector would be used for gene targeting in ES cells in efforts to generate TetRkid knock-in mouse.